Amino acid-, peptide- and polypeptide-lipids, isomers, compositions, and uses thereof

ABSTRACT

wherein m, n, p, R′, R1, R2, R3, R4, R5, R8, Z, W, Y, and Z are as defined herein.

RELATED APPLICATIONS

The present application is a division of U.S. application Ser. No.15/340,082, filed Nov. 1, 2016, which is a division of and claimspriority under 35 U.S.C. § 120 to U.S. application Ser. No. 13/662,002,filed Oct. 26, 2012, which claims priority under 35 U.S.C. § 119(e) toU.S. provisional patent application, U.S. Ser. No. 61/552,423, filedOct. 27, 2011, each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. R37EB000244 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

The ability to silence genes via RNA interference (RNAi) was reported byMello and Fire in 1998. See Fire et al., Nature (1998) 391:806-811.Since then, scientists have rushed to take advantage of the enormoustherapeutic potential driven by targeted gene knockdown. This isevidenced by the fact that the first report of small interfering RNA(siRNA) mediated RNAi in human beings was reported only twelve yearsafter the phenomenon was described in Caenorhabditis elegans. See Daviset al., Nature (2010) 464:1067-1070. It is well understood thatdevelopment of genetic drugs is slowed by the inability to delivernucleic acids effectively in vivo. When unprotected, genetic materialinjected into the bloodstream can be degraded by DNAases and RNAases,or, if not degraded, the genetic material can stimulate an immuneresponse. See, e.g., Whitehead et al., Nature Reviews Drug Discovery(2009) 8:129-138; Robbins et al., Oligonucleotides (2009) 19:89-102.Intact siRNA must then enter the cytosol, where the antisense strand isincorporated into the RNA-induced silencing complex (RISC) (Whiteheadsupra). The RISC associates with and degrades complementary mRNAsequences, thereby preventing translation of the target mRNA intoprotein, i.e., “silencing” the gene.

To overcome difficulties in delivery, nucleotides have been complexedwith a wide variety of delivery systems, including polymers, lipids,inorganic nanoparticles and viruses. See, e.g., Peer et al. NatureNanotechnology, (2007) 2:751-760. However, despite promising data fromongoing clinical trials for the treatment of respiratory syncytial virusand liver cancers (see, e.g., Zamora et al., Am. J. Respir. Crit. CareMed. (2011) 183:531-538), the clinical use of siRNA continues to requiredevelopment of safer and more effective delivery systems. Toward thisend, numerous lipid-like molecules have been developed including polyβ-amino esters and amino alcohol lipids. See, e.g., PCT ApplicationPublication Nos. WO 2002/031025; WO 2004/106411; WO 2008/011561; WO2007/143659; WO 2006/138380; and WO 2010/053572. Amino acid, peptide,polypeptide-lipids (APPL) have also been studied for a variety ofapplications, including use as therapeutics, biosurfactants, andnucleotide delivery systems. See, e.g., Giuliani et al., Cellular andMolecular Life Sciences (2011) 68:2255-2266; Ikeda et al., CurrentMedicinal Chemistry (2007) 14: 111263-1275; Sen, Advances inExperimental Medicine and Biology (2010) 672:316-323; and Damen et al.,Journal of Controlled Release (2010) 145:33-39. However, there continuesto remain a need to investigate and develop new APPL systems withimproved properties, such as new and improved APPL nucleotide deliverysystems.

SUMMARY OF THE INVENTION

Described herein are inventive compounds and compositions characterized,in certain embodiments, by conjugation of various groups, such aslipophilic groups, to an amino or amide group of an amino acid, a linearor cyclic peptide, a linear or cyclic polypeptide, or structural isomerthereof, to provide compounds of the present invention, collectivelyreferred to herein as “APPLs”. Such APPLs are deemed useful for avariety of applications, such as, for example, improved nucleotidedelivery.

Exemplary APPLs include, but are not limited to, compounds of Formula(I), (II), (III), (IV), (V), and (VI), and salts thereof, as describedherein:

wherein m, n, p, R′, R¹, R², R³, R⁴, R⁵, R⁸, Z, W, Y, and Z are asdefined herein, provided that the APPL comprises at least one instanceof a group of formula (i), (ii), or (iii):

wherein:

each instance of R′ is independently hydrogen or optionally substitutedalkyl;

X is O, S, NR^(X), wherein R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

Y is O, S, NR^(Y), wherein R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

R^(P) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached to asulfur atom, or a nitrogen protecting group when attached to a nitrogenatom; and

R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substitutedC₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionallysubstituted heteroC₁₋₅₀alkyl, optionally substituted heteroC₂₋₅₀alkenyl,optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.

In certain embodiments, the group of formula (i) represents a group offormula (i-a) or a group of formula (i-b):

In certain embodiments, the group of formula (i-a) is a group of formula(i-a1) or a group of formula (i-a2):

In certain embodiments, the group of formula (i-b) is a group of formula(i-b1) or a group of formula (i-b2):

In certain embodiments, at least one instance of R¹ is a group offormula:

wherein L is an optionally substituted alkylene, optionally substitutedalkenylene, optionally substituted alkynylene, optionally substitutedheteroalkylene, optionally substituted heteroalkenylene, optionallysubstituted heteroalkynylene, optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,or optionally substituted heteroarylene, and

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, and a nitrogen protecting group;

provided at least one instance of R⁶ and R⁷ is a group of formula:

wherein:

each instance of R′ is independently hydrogen or optionally substitutedalkyl;

X is O, S, NR^(X), wherein R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

Y is O, S, NR^(Y), wherein R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

R^(P) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached to asulfur atom, or a nitrogen protecting group when attached to a nitrogenatom; and

R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substitutedC₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionallysubstituted heteroC₁₋₅₀alkyl, optionally substituted heteroC₂₋₅₀alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.

In certain embodiments, each instance of R′ is hydrogen.

In certain embodiments, L is an optionally substituted alkylene.

In certain embodiments, the group of formula (iv) is of formula:

wherein q is an integer between 1 and 50, inclusive.

In certain embodiments, each instance of R¹ is a group of formula (iv).

An exemplary APPL of the present invention is compound (cKK-E12):

or a salt thereof.

In another aspect, provided are compositions comprising an APPL or asalt thereof.

For example, in certain embodiments, provided is a compositioncomprising an APPL or salt thereof and, optionally, an excipient,wherein the APPL is an amino acid, a linear or cyclic peptide, a linearor cyclic polypeptide, or structural isomer thereof, and wherein anamino or amide group of the APPL is conjugated to a group of formula(i), (ii), or (iii). In certain embodiments, the group of formula (i),(ii), or (iii) is attached to an amino group present on the APPLscaffold. In certain embodiments, the composition is a pharmaceuticalcomposition, a cosmetic composition, a nutraceutical composition, or acomposition with non-medical application. In certain embodiments, thecomposition with non-medical application is an emulsion or emulsifieruseful as a food component, for extinguishing fires, for disinfectingsurfaces, or for oil cleanup.

In certain embodiments, the composition further comprises an agent. Incertain embodiments, the agent is an organic molecule, inorganicmolecule, nucleic acid, protein, peptide, polynucleotide, targetingagent, an isotopically labeled chemical compound, vaccine, animmunological agent, or an agent useful bioprocessing, e.g., in theintracellular manufacturing of proteins. In certain embodiments, theagent is a polynucleotide, and the polynucleotide is DNA or RNA. Incertain embodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, orantisense RNA. In certain embodiments, the agent and the APPL are notcovalently attached, e.g., for example, the agent and the APPL arenon-covalently complexed to each other. However, in certain embodiments,the agent and the APPL are covalently attached.

In certain embodiments, the composition is in the form of a particle. Incertain embodiments, the particle is a nanoparticle or microparticle. Incertain embodiments, the particle is a micelle, liposome, or lipoplex.In certain embodiments, the particle encapsulates an agent, e.g., anagent to be delivered.

In another aspect, provided is a method of delivering a polynucleotideto a biological cell, comprising providing a composition comprising anAPPL, or salt thereof, and a polynucleotide, and exposing thecomposition to the biological cell under conditions sufficient tofacilitate delivery of the polynucleotide into the interior of thebiological cell; wherein the APPL is an amino acid, a linear or cyclicpeptide, or a linear or cyclic polypeptide, or structural isomerthereof, wherein an amino or amide group of the APPL is conjugated to agroup of formula (i), (ii), or (iii). In certain embodiments, thepolynucleotide is DNA or RNA. In certain embodiments, the RNA is RNAi,dsRNA, siRNA, shRNA, miRNA, or antisense RNA. In certain embodiments,upon delivery of the RNA into the cell, the RNA is able to interferewith the expression of a specific gene in the biological cell.

In yet another aspect, provided are screening methods. For example, inone embodiment, provided is a method of screening a compound library,the method comprising providing a plurality of different APPLs, or saltsthereof, and performing at least one assay with the compound library todetermine the presense or absence of a desired property; wherein theAPPL is an amino acid, a linear or cyclic peptide, or a linear or cyclicpolypeptide, or structural isomer thereof, wherein an amino or amidegroup of the APPL is conjugated to a group of formula (i), (ii), or(iii). In certain embodiments, the desired property is solubility inwater, solubility at different pH, ability to bind polynucleotides,ability to bind heparin, ability to bind small molecules, ability tobind protein, ability to form microparticles, ability to increasetranfection efficiency, ability to support cell growth, ability tosupport cell attachment, ability to support tissue growth, and/orintracellular delivery of the APPL and/or an agent complexed or attachedthereto to aid in bioprocessing.

In still yet another aspect, provided are methods of use of theinventive APPLs for the treatment of various diseases, disorders, orconditions. For example, in certain embodiments, provided is a method oftreating a disease, disorder, or condition from which the subjectsuffers, comprising administering to a subject in need thereof aneffective amount of an APPL, or salt thereof, wherein the APPL is anamino acid, a linear or cyclic peptide, or a linear or cyclicpolypeptide, or structural isomer thereof, wherein an amino or amidegroup of the APPL is conjugated to a group of formula (i), (ii), or(iii).

The details of one or more embodiments of the invention are set forthherein. Other features, objects, and advantages of the invention will beapparent from the Detailed Description, the Figures, the Examples, andthe Claims.

Definitions Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L.Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen,S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L.Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). Theinvention additionally encompasses compounds as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

As used herein, “alkyl” refers to a radical of a straight-chain orbranched saturated hydrocarbon group having from 1 to 50 carbon atoms(“C₁₋₅₀alkyl”). In some embodiments, an alkyl group has 1 to 40 carbonatoms (“C₁₋₄ alkyl”). In some embodiments, an alkyl group has 1 to 30carbon atoms (“C₁₋₃₀ alkyl”). In some embodiments, an alkyl group has 1to 20 carbon atoms (“C₁₋₂₀ alkyl”). In some embodiments, an alkyl grouphas 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkylgroup has 1 to 9 carbon atoms (“C₁₋₉ alkyl”). In some embodiments, analkyl group has 1 to 8 carbon atoms (“C₁₋₈ alkyl”). In some embodiments,an alkyl group has 1 to 7 carbon atoms (“C₁₋₇ alkyl”). In someembodiments, an alkyl group has 1 to 6 carbon atoms (“C₁₋₆ alkyl”). Insome embodiments, an alkyl group has 1 to 5 carbon atoms (“C₁₋₅ alkyl”).In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C₁₋₄alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms(“C₁₋₃ alkyl”). In some embodiments, an alkyl group has 1 to 2 carbonatoms (“C₁₋₂ alkyl”). In some embodiments, an alkyl group has 1 carbonatom (“C₁ alkyl”). In some embodiments, an alkyl group has 2 to 6 carbonatoms (“C₂₋₆ alkyl”). Examples of C₁₋₆ alkyl groups include methyl (C₁),ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl(C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅),amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅),and n-hexyl (C₆). Additional examples of alkyl groups include n-heptyl(C₇), n-octyl (C₈) and the like. Unless otherwise specified, eachinstance of an alkyl group is independently unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents. In certain embodiments, the alkyl group is anunsubstituted C₁₋₅₀alkyl. In certain embodiments, the alkyl group is asubstituted C₁₋₅₀alkyl.

As used herein, “heteroalkyl” refers to an alkyl group as defined hereinwhich further includes at least one heteroatom (e.g., 1 to 25, e.g., 1,2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron,silicon, or phosphorus within (i.e., inserted between adjacent carbonatoms of) and/or placed at one or more terminal position(s) of theparent chain. In certain embodiments, a heteroalkyl group refers to asaturated group having from 1 to 50 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC₁₋₅₀alkyl”). In certainembodiments, a heteroalkyl group refers to a saturated group having from1 to 40 carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₄₀ alkyl”). In certain embodiments, a heteroalkyl grouprefers to a saturated group having from 1 to 30 carbon atoms and 1 ormore heteroatoms within the parent chain (“heteroC₁₋₃₀alkyl”). Incertain embodiments, a heteroalkyl group refers to a saturated grouphaving from 1 to 20 carbon atoms and 1 or more heteroatoms within theparent chain (“heteroC₁₋₂₀alkyl”). In certain embodiments, a heteroalkylgroup refers to a saturated group having from 1 to 10 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₁₀alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC₁₋₅alkyl”). In some embodiments, a heteroalkylgroup is a saturated group having 1 to 4 carbon atoms and 1 or 2heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₅₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC₁₋₅₀ alkyl.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 50 carbon atoms and one ormore carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds)(“C₂₋₅₀ alkenyl”). In some embodiments, an alkenyl group has 2 to 40carbon atoms (“C₂₋₄₀ alkenyl”). In some embodiments, an alkenyl grouphas 2 to 30 carbon atoms (“C₂₋₃₀ alkenyl”). In some embodiments, analkenyl group has 2 to 20 carbon atoms (“C₂₋₂₀ alkenyl”). In someembodiments, an alkenyl group has 2 to 10 carbon atoms (“C₂₋₁₀alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms(“C₂₋₉ alkenyl”). In some embodiments, an alkenyl group has 2 to 8carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, analkenyl group has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In someembodiments, an alkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”).In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂alkenyl”). The one or more carbon-carbon double bonds can be internal(such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples ofC₂₋₄ alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl(C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like.Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkenylgroups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and thelike. Additional examples of alkenyl include heptenyl (C₇), octenyl(C₈), octatrienyl (C₈), and the like. Unless otherwise specified, eachinstance of an alkenyl group is independently unsubstituted (an“unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) withone or more substituents. In certain embodiments, the alkenyl group isan unsubstituted C₂₋₅₀ alkenyl. In certain embodiments, the alkenylgroup is a substituted C₂₋₅₀ alkenyl.

As used herein, “heteroalkenyl” refers to an alkenyl group as definedherein which further includes at least one heteroatom (e.g., 1 to 25,e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen,boron, silicon, or phosphorus within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkenyl group refersto a group having from 2 to 50 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₅₀alkenyl”). In certain embodiments, a heteroalkenyl group refers to agroup having from 2 to 40 carbon atoms, at least one double bond, and 1or more heteroatoms within the parent chain (“heteroC₂₋₄₀ alkenyl”). Incertain embodiments, a heteroalkenyl group refers to a group having from2 to 30 carbon atoms, at least one double bond, and 1 or moreheteroatoms within the parent chain (“heteroC₂₋₃₀ alkenyl”). In certainembodiments, a heteroalkenyl group refers to a group having from 2 to 20carbon atoms, at least one double bond, and 1 or more heteroatoms withinthe parent chain (“heteroC₂₋₂₀ alkenyl”). In certain embodiments, aheteroalkenyl group refers to a group having from 2 to 10 carbon atoms,at least one double bond, and 1 or more heteroatoms within the parentchain (“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenylgroup has 2 to 9 carbon atoms at least one double bond, and 1 or moreheteroatoms within the parent chain (“heteroC₂₋₉ alkenyl”). In someembodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₈ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 7 carbon atoms, at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”).In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₅₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₅₀ alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 50 carbon atoms and one ormore carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) andoptionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds)(“C₂₋₅₀ alkynyl”). An alkynyl group that has one or more triple bondsand one or more double bonds is also referred to as an “ene-yene”. Insome embodiments, an alkynyl group has 2 to 40 carbon atoms (“C₂₋₄₀alkynyl”). In some embodiments, an alkynyl group has 2 to 30 carbonatoms (“C₂₋₃₀ alkynyl”). In some embodiments, an alkynyl group has 2 to20 carbon atoms (“C₂₋₂₀ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 10 carbon atoms (“C₂₋₁₀ alkynyl”). In some embodiments, analkynyl group has 2 to 9 carbon atoms (“C₂₋₉ alkynyl”). In someembodiments, an alkynyl group has 2 to 8 carbon atoms (“C₂₋₈ alkynyl”).In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C₂₋₇alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms(“C₂₋₆ alkynyl”). In some embodiments, an alkynyl group has 2 to 5carbon atoms (“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has2 to 4 carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynylgroup has 2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, analkynyl group has 2 carbon atoms (“C₂ alkynyl”). The one or morecarbon-carbon triple bonds can be internal (such as in 2-butynyl) orterminal (such as in 1-butynyl). Examples of C₂₋₄ alkynyl groupsinclude, without limitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl(C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkynyl groups as well aspentynyl (C₅), hexynyl (C₆), and the like. Additional examples ofalkynyl include heptynyl (C₇), octynyl (C₈), and the like. Unlessotherwise specified, each instance of an alkynyl group is independentlyunsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is an unsubstituted C₂₋₅₀ alkynyl. Incertain embodiments, the alkynyl group is a substituted C₂₋₅₀ alkynyl.

As used herein, “heteroalkynyl” refers to an alkynyl group as definedherein which further includes at least one heteroatom (e.g., 1 to 25,e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen,boron, silicon, or phosphorus within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkynyl group refersto a group having from 2 to 50 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₅₀alkynyl”). In certain embodiments, a heteroalkynyl group refers to agroup having from 2 to 40 carbon atoms, at least one triple bond, and 1or more heteroatoms within the parent chain (“heteroC₂₋₄₀ alkynyl”). Incertain embodiments, a heteroalkynyl group refers to a group having from2 to 30 carbon atoms, at least one triple bond, and 1 or moreheteroatoms within the parent chain (“heteroC₂₋₃₀ alkynyl”). In certainembodiments, a heteroalkynyl group refers to a group having from 2 to 20carbon atoms, at least one triple bond, and 1 or more heteroatoms withinthe parent chain (“heteroC₂₋₂₀ alkynyl”). In certain embodiments, aheteroalkynyl group refers to a group having from 2 to 10 carbon atoms,at least one triple bond, and 1 or more heteroatoms within the parentchain (“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynylgroup has 2 to 9 carbon atoms, at least one triple bond, and 1 or moreheteroatoms within the parent chain (“heteroC₂₋₉ alkynyl”). In someembodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₈ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 7 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”).In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₅₀alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC₂₋₅₀ alkynyl.

As used herein, “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbonatoms (“C₃₋₁₀ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 7 ring carbon atoms (“C₃₋₇ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ carbocyclyl”). In some embodiments, a carbocyclylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ carbocyclyl”). In someembodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include, withoutlimitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl (C₄),cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅), cyclohexyl(C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like. ExemplaryC₃₋₈ carbocyclyl groups include, without limitation, the aforementionedC₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇), cycloheptenyl (C₇),cycloheptadienyl (C₇), cycloheptatrienyl (C₇), cyclooctyl (C₈),cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇), bicyclo[2.2.2]octanyl(C₈), and the like. Exemplary C₃₋₁₀ carbocyclyl groups include, withoutlimitation, the aforementioned C₃₋₈ carbocyclyl groups as well ascyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀), cyclodecenyl(C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl (C₁₀),spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₀carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₀ carbocyclyl.

In some embodiments, “carbocyclyl” or “carbocyclic” is referred to as a“cycloalkyl”, i.e., a monocyclic, saturated carbocyclyl group havingfrom 3 to 10 ring carbon atoms (“C₃₋₁₀ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C₃₋₈cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ringcarbon atoms (“C₃₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 4 to 6 ring carbon atoms (“C₄₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is an unsubstituted C₃₋₁₀ cycloalkyl.In certain embodiments, the cycloalkyl group is a substituted C₃₋₁₀cycloalkyl.

As used herein, “heterocyclyl” or “heterocyclic” refers to a radical ofa 3- to 14-membered non-aromatic ring system having ring carbon atomsand 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein eachheteroatom is independently selected from oxygen, sulfur, nitrogen,boron, silicon, or phosphorus (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1 or more (e.g.,1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom isindependently selected from oxygen, sulfur, nitrogen, boron, silicon, orphosphorus (“5-10 membered heterocyclyl”). In some embodiments, aheterocyclyl group is a 5-8 membered non-aromatic ring system havingring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms,wherein each heteroatom is independently selected from oxygen, sulfur,nitrogen, boron, silicon, or phosphorus (“5-8 membered heterocyclyl”).In some embodiments, a heterocyclyl group is a 5-6 membered non-aromaticring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4)ring heteroatoms, wherein each heteroatom is independently selected fromoxygen, sulfur, nitrogen, boron, silicon, or phosphorus (“5-6 memberedheterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen,sulfur, nitrogen, boron, silicon, or phosphorus. In some embodiments,the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected fromoxygen, sulfur, nitrogen, boron, silicon, or phosphorus. In someembodiments, the 5-6 membered heterocyclyl has 1 ring heteroatomselected from oxygen, sulfur, nitrogen, boron, silicon, or phosphorus.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary4-membered heterocyclyl groups containing 1 heteroatom include, withoutlimitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-memberedheterocyclyl groups containing 1 heteroatom include, without limitation,tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl,dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione.Exemplary 5-membered heterocyclyl groups containing 2 heteroatomsinclude, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.Exemplary 5-membered heterocyclyl groups containing 3 heteroatomsinclude, without limitation, triazolinyl, oxadiazolinyl, andthiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1heteroatom include, without limitation, piperidinyl, tetrahydropyranyl,dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, piperazinyl,morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclylgroups containing 2 heteroatoms include, without limitation,triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1heteroatom include, without limitation, azepanyl, oxepanyl andthiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

As used herein, “aryl” refers to a radical of a monocyclic or polycyclic(e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6,10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbonatoms and zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

As used herein, “heteroaryl” refers to a radical of a 5-14 memberedmonocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromaticring system (e.g., having 6, 10, or 14 π electrons shared in a cyclicarray) having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4 ringheteroatoms) ring heteroatoms provided in the aromatic ring system,wherein each heteroatom is independently selected from oxygen, sulfur,nitrogen, boron, silicon, or phosphorus (“5-14 membered heteroaryl”). Inheteroaryl groups that contain one or more nitrogen atoms, the point ofattachment can be a carbon or nitrogen atom, as valency permits.Heteroaryl polycyclic ring systems can include one or more heteroatomsin one or both rings. “Heteroaryl” includes ring systems wherein theheteroaryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the point of attachment is on theheteroaryl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heteroaryl ringsystem. “Heteroaryl” also includes ring systems wherein the heteroarylring, as defined above, is fused with one or more aryl groups whereinthe point of attachment is either on the aryl or heteroaryl ring, and insuch instances, the number of ring members designates the number of ringmembers in the fused polycyclic (aryl/heteroaryl) ring system.Polycyclic heteroaryl groups wherein one ring does not contain aheteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) thepoint of attachment can be on either ring, i.e., either the ring bearinga heteroatom (e.g., 2-indolyl) or the ring that does not contain aheteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from oxygen, sulfur, nitrogen,boron, silicon, or phosphorus (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from oxygen, sulfur, nitrogen,boron, silicon, or phosphorus (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from oxygen, sulfur, nitrogen,boron, silicon, or phosphorus (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1 or more (e.g., 1, 2, or3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron,silicon, or phosphorus. In some embodiments, the 5-6 membered heteroarylhas 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen,boron, silicon, or phosphorus. In some embodiments, the 5-6 memberedheteroaryl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen,boron, silicon, or phosphorus. Unless otherwise specified, each instanceof a heteroaryl group is independently unsubstituted (an “unsubstitutedheteroaryl”) or substituted (a “substituted heteroaryl”) with one ormore substituents. In certain embodiments, the heteroaryl group is anunsubstituted 5-14 membered heteroaryl. In certain embodiments, theheteroaryl group is a substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups (e.g., arylor heteroaryl moieties) as herein defined.

As used herein, the term “saturated” refers to a ring moiety that doesnot contain a double or triple bond, i.e., the ring contains all singlebonds.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

As understood from the above, alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl groups, as defined herein, are, in certain embodiments,optionally substituted. Optionally substituted refers to a group whichmay be substituted or unsubstituted (e.g., “substituted” or“unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl,“substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” heteroalkyl, “substituted” or “unsubstituted”heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted” means that at least one hydrogen present on a group isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein that results in theformation of a stable compound. The present invention contemplates anyand all such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SeH, —SeR^(aa), —SH,—SR^(aa), —SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂,—CO₂R^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂,—OC(═O)N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—OC(═NR^(bb))N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—C(═O)NR^(bb)SO₂R^(aa), —NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa),—SO₂OR^(aa), —OSO₂R^(aa), —S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₄carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₅₀alkyl,C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(aa)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(bb) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(cc)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₅₀ alkyl, C₂₋₅₀alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or twogeminal R^(dd) substituents can be joined to form ═O or ═S;

each instance of R^(ee) is, independently, selected from C₁₋₅₀alkyl,C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups;

each instance of R^(ff) is, independently, selected from hydrogen,C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-10membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, or twoR^(ff) groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₅₀ alkyl, —ON(C₁₋₅₀alkyl)₂, —N(C₁₋₅₀ alkyl)₂,—N(C₁₋₅₀ alkyl)₃ ⁺X⁻, —NH(C₁₋₅₀ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₅₀ alkyl)+X⁻, —NH₃⁺X⁻, —N(OC₁₋₅₀ alkyl)(C₁₋₅₀ alkyl), —N(OH)(C₁₋₅₀ alkyl), —NH(OH), —SH,—SC₁₋₅₀ alkyl, —SS(C₁₋₅₀ alkyl), —C(═O)(C₁₋₅₀ alkyl), —CO₂H, —CO₂(C₁₋₅₀alkyl), —OC(═O)(C₁₋₅₀ alkyl), —OCO₂(C₁₋₅₀ alkyl), —C(═O)NH₂,—C(═O)N(C₁₋₅₀ alkyl)₂, —OC(═O)NH(C₁₋₅₀ alkyl), —NHC(═O)(C₁₋₅₀alkyl),—N(C₁₋₅₀ alkyl)C(═O)(C₁₋₅₀ alkyl), —NHCO₂(C₁₋₅₀ alkyl), —NHC(═O)N(C₁₋₅₀alkyl)₂, —NHC(═O)NH(C₁₋₅₀ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₅₀alkyl),—OC(═NH)(C₁₋₅₀ alkyl), —OC(═NH)OC₁₋₅₀ alkyl, —C(═NH)N(C₁₋₅₀ alkyl)₂,—C(═NH)NH(C₁₋₅₀ alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₅₀ alkyl)₂,—OC(NH)NH(C₁₋₅₀ alkyl), —OC(NH)NH₂, —NHC(NH)N(C₁₋₅₀ alkyl)₂,—NHC(═NH)NH₂, —NHSO₂(C₁₋₅₀ alkyl), —SO₂N(C₁₋₅₀ alkyl)₂, —SO₂NH(C₁₋₅₀alkyl), —SO₂NH₂, —SO₂C₁₋₅₀ alkyl, —SO₂OC₁₋₅₀ alkyl, —OSO₂C₁₋₅₀ alkyl,—SOC₁₋₆ alkyl, —Si(C₁₋₅₀ alkyl)₃, —OSi(C₁₋₆ alkyl)₃ —C(═S)N(C₁₋₅₀alkyl)₂, C(═S)NH(C₁₋₅₀ alkyl), C(═S)NH₂, —C(═O)S(C₁₋₆ alkyl),—C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₅₀ alkyl),—P(═O)(C₁₋₅₀ alkyl)₂, —OP(═O)(C₁₋₅₀ alkyl)₂, —OP(═O)(OC₁₋₅₀ alkyl)₂,C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or twogeminal R^(gg) substituents can be joined to form ═O or ═S;

wherein X⁻ is a counterion.

As used herein, the term “halo” or “halogen” refers to fluorine (fluoro,—F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

As used herein, a “counterion” is a negatively charged group associatedwith a positively charged quarternary amine in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(aa), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(cc) groups attached to an N atom are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, andwherein R^(aa), R^(bb), R^(cc) and R^(dd) are as defined above.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(aa),—SO₂OR^(aa), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc) and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, o-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, co-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is ansulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —R^(aa),—N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. Sulfur protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

As used herein, a “leaving group” is an art-understood term referring toa molecular fragment that departs with a pair of electrons inheterolytic bond cleavage, wherein the molecular fragment is an anion orneutral molecule. See, for example, Smith, March Advanced OrganicChemistry 6th ed. (501-502). Exemplary leaving groups include, but arenot limited to, halo (e.g., chloro, bromo, iodo) and sulfonylsubstituted hydroxyl groups (e.g., tosyl, mesyl, besyl).

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, Figures, and Claims. The inventionis not intended to be limited in any manner by the above exemplarylisting of substituents.

Other Definitions

As used herein, use of the phrase “at least one instance” refers to oneinstance, but also encompasses more than one instance, e.g., forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 instances, and up to 100instances.

An “amino acid” refers to natural and unnatural D/L alpha-amino acids,as well as natural and unnatural beta- and gamma-amino acids. A“peptide” refers to two amino acids joined by a peptide bond. A“polypeptide” refers to three or more amino acids joined by peptidebonds. An “amino acid side chain” refers to the group(s) pended to thealpha carbon (if an alpha amino acid), alpha and beta carbon (if a betaamino acid), or the alpha, beta, and gamma carbon (if a gamma aminoacid). Exemplary amino acid side chains are depicted herein; see, e.g.,Table 1 of the Examples.

As used herein, a “polymer” refers to a compound comprised of at least 3(e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeatingcovalently bound structural units.

“Conjugated” and “attached” refer to the covalent attachment of a group,and are used interchangeably herein.

As used herein, “lipophilic” refers to the ability of a group todissolve in fats, oils, lipids, and lipophilic non-polar solvents suchas hexane or toluene. In general, a lipophilic group refers to anunsubstituted n-alkyl or unsubstituted n-alkenyl group having 6 to 50carbon atoms, e.g., 6 to 40, 6 to 30, 6 to 20, 8 to 20, 8 to 19, 8 to18, 8 to 17, 8 to 16, or 8 to 15 carbon atoms.

Use of the terms “structural isomer,” “organic molecule,” and “inorganicmolecule” are meant to encompass the common meaning of each term asknown in the art.

As used herein, a “small organic molecule” or “small molecule” refers toan organic molecule with a molecular weight of 800 g/mol or less (e.g.,less than 700 g/mol, less than 600 g/mol, less than 500 g/mol, less than400 g/mol, less than 300 g/mol, less than 200 g/mol, less than 100g/mol, between 50 to 800 g/mol, inclusive, between 100 to 800 g/mol,inclusive, or between 100 to 500 g/mol, inclusive). In certainembodiments, the small organic molecule is a therapeutically activeagent such as a drug (e.g., a small organic molecule approved by theU.S. Food and Drug Administration as provided in the Code of FederalRegulations (CFR)). The small organic molecule may also be complexedwith a metal. In this instance, the small organic molecule is alsoreferred to as an “small organometallic molecule.”

As used herein, a “large organic molecule” or “large molecule” refers toan organic compound with a molecular weight of greater than 800 g/mol(e.g., greater than 800 g/mol, greater than 900 g/mol, greater than 1000g/mol, greater than 2000 g/mol, between 801 to 2000 g/mol, inclusive,between 900 to 2000 g/mol, inclusive, between 1000 to 2000 g/mol,inclusive, or between 801 to 1000 g/mol, inclusive). In certainembodiments, the large organic molecule is a therapeutically activeagent such as a drug (e.g., a large organic molecule approved by theU.S. Food and Drug Administration as provided in the Code of FederalRegulations (CFR)). The large organic molecule may also be complexedwith a metal. In this instance, the large organic molecule is alsoreferred to as an “large organometallic compound.”

As used herein, a “small inorganic molecule” refers to an inorganiccompound with a molecular weight of 800 g/mol or less (e.g., less than700 g/mol, less than 600 g/mol, less than 500 g/mol, less than 400g/mol, less than 300 g/mol, less than 200 g/mol, less than 100 g/mol,between 50 to 800 g/mol, inclusive, between 100 to 800 g/mol, inclusive,or between 100 to 500 g/mol, inclusive). In certain embodiments, thesmall inorganic molecule is a therapeutically active agent such as adrug (e.g., a small inorganic molecule approved by the U.S. Food andDrug Administration as provided in the Code of Federal Regulations(CFR)).

As used herein, a “large inorganic molecule” refers to an inorganiccompound with a molecular weight of greater than 800 g/mol (e.g.,greater than 800 g/mol, greater than 900 g/mol, greater than 1000 g/mol,greater than 2000 g/mol, between 801 to 2000 g/mol, inclusive, between900 to 2000 g/mol, inclusive, between 1000 to 2000 g/mol, inclusive, orbetween 801 to 1000 g/mol, inclusive). In certain embodiments, the largeinorganic molecule is a therapeutically active agent such as a drug(e.g., a large inorganic molecule approved by the U.S. Food and DrugAdministration as provided in the Code of Federal Regulations (CFR)).

As used herein, the term “salt” or “pharmaceutically acceptable salt”refers to those salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describes pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceuticallyacceptable salts of the compounds of this invention include thosederived from suitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Further pharmaceutically acceptable salts include salts formed from thequarternization of an amine using an appropriate electrophile, e.g., analkyl halide, to form a quarternized alkylated amino salt.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structural design and optimization through in vivoevaluation in mice. Single amino acid-based lipid derivatives weretested at a dose of 1 mg/kg in mice, which indicated that lysine was afavorable amino acid. Lysine-based peptide and polypeptide-lipidderivatives were then investigated at the same dose. The hit rate wasimproved from 1.7% to 23% (including those compounds not screened due toparticle instability or no entrapment of siRNA). The top hits and theiranalogs were explored at a lower dose of 0.1 mg/kg, which led toselection of cKK-E12 as the lead compound. K-E12; K: abbreviation oflysine, E: epoxide, A: aldehyde, O: acrylate, 12: carbon tail length.cKK-E12; c: cyclic; Control, phosphate-buffered saline.

FIG. 2 depicts the bio-distribution of free Cy5.5-labeled siRNA andCy5.5-labeled siRNA-cKK-E12 formulation in mice at 1 hr and 24 hr.

FIG. 3 depicts the silencing effects of apolipoproteins on cKK-E12 inHeLa cells. Apolipoproteins including ApoA-I (recombinant Human ApoA-Iprotein), ApoA-II (native Human ApoA-II protein), ApoB (native HumanApoB protein), ApoC-I (native Human ApoC-I protein), ApoC-II (nativeHuman ApoC-II protein), ApoC-III (native Human ApoC-III protein), ApoE(native Human ApoE protein), ApoE2 (recombinant Human ApoE2 protein),ApoE3 (recombinant Human ApoE3 protein), ApoE4 (recombinant Human ApoE4protein), ApoH (native Human ApoH protein).

FIG. 4 depicts the effects of ApoE on gene silencing and cell uptake.A). Silencing effects of ApoE on cKK-E12, cKK-A12, and cKK-O12 in vitro(siRNA: 50 ng/well). With addition of ApoE, the order of silencingeffects was cKK-E12>cKK-A12>cKK-O12, correlating well with in vivoactivity. B). Cellular internalization of cKK-E12 with Alex-647 labeledsiRNA after 3 hr of incubation is demonstrated by HT automated confocalmicroscopy. ApoE enhanced cell uptake and endosomal escape of cKK-E12;Scale bar: 20 μm.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Described herein are inventive compounds and compositions, certainembodiments of which involve conjugation of various groups, such aslipophilic groups, to an amino or amide group of an amino acid, a linearor cyclic peptide, a linear or cyclic polypeptide, or structural isomerthereof, to provide compounds of the present invention, collectivelyreferred to herein as “APPLs”. Such APPLs are deemed useful for avariety of applications, such as, for example, improved nucleotidedelivery.

Exemplary APPLs include, but are not limited to, compounds of Formula(I), (II), (III), (IV), (V), and (VI), and salts thereof, as describedherein:

wherein m, n, p, R¹, R², R³, R⁴, R⁵, R⁸, Z, W, Y, and Z are as definedherein.

Various R^(L) groups, e.g., lipophilic groups, may be attached to theAPPL via conjugation of a primary or secondary amino group or amide ofthe amino acid, peptide, or polypeptide precursor, or structural isomerthereof, with an epoxide, thiirane, or aziridine of formula (i-x),Michael addition to an α,β-unsaturated ester, thioester, or amide offormula (ii-x), or reductive amination to an aldehyde of formula (iii-x)(Scheme 1).

Thus, in the broadest aspect, the present invention provides APPLs, andin certain embodiments, compounds of Formula (I), (II), (III), (IV),(V), and (VI), comprising at least one instance of a group attachedthereto of the formula:

wherein:

each instance of R′ is independently hydrogen or optionally substitutedalkyl;

X is O, S, NR^(X), wherein R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

Y is O, S, NR^(Y), wherein R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

R^(P) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached to asulfur atom, or a nitrogen protecting group when attached to a nitrogenatom; and

R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substitutedC₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionallysubstituted C₁₋₅₀ heteroalkyl, optionally substituted C₂₋₅₀heteroalkenyl, optionally substituted C₂₋₅₀ heteroalkynyl, or a polymer.

Various embodiments of formula (i), (ii), and (iii), and variablesR^(L), R^(P), X, and Y are described in greater detail herein.

Compounds of Formula (I)

Compounds of Formula (I) encompasses amino acids, linear peptides, andlinear polypeptides which comprise one or more sites of conjugation,e.g., to the terminal amino group, to an amino substituent, and/or to animino nitrogen, of a group of formula (i), (ii), or (iii).

Thus, in one aspect, provided is a compound of Formula (I):

or salt thereof;wherein:

n is 0 or is an integer between 1 and 100,000, inclusive;

each instance of m is independently 1, 2, or 3;

each instance of Z is independently O, S, or NR^(Z), wherein R^(Z) ishydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group, or agroup of the formula (i), (ii), or (iii);

each instance of R¹ is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, halogen,—OR^(A1), —N(R^(A1))₂, or —SR^(A1); wherein each occurrence of R^(A1) isindependently hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, an oxygenprotecting group when attached to an oxygen atom, a sulfur protectinggroup when attached to an sulfur atom, a nitrogen protecting group whenattached to a nitrogen atom, or two R^(A1) groups are joined to form anoptionally substituted heterocyclic or optionally substituted heteroarylring;

R² is a group of formula (i), (ii), or (iii);

R³ is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, a nitrogen protecting group, ora group of the formula (i), (ii), or (iii);

or R³ and an R¹ group are joined to form an optionally substituted 5-6membered heterocyclic ring;

R⁴ is —OR^(A4), —N(R^(A4))₂, or —SR^(A4); wherein each occurrence ofR^(A4) is independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to an oxygen atom, a sulfurprotecting group when attached to an sulfur atom, a nitrogen protectinggroup when attached to a nitrogen atom, or two R^(A4) groups are joinedto form an optionally substituted heterocyclic or optionally substitutedheteroaryl ring;

R⁵ is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, or a nitrogen protecting group;and

Formulae (i), (ii), and (iii) are:

wherein:

each instance of R′ is independently hydrogen or optionally substitutedalkyl;

X is O, S, NR^(X), wherein R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

Y is O, S, NR^(Y), wherein R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

R^(P) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached to asulfur atom, or a nitrogen protecting group when attached to a nitrogenatom; and

R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substitutedC₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionallysubstituted heteroC₁₋₅₀alkyl, optionally substituted heteroC₂₋₅₀alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.

In certain embodiments, when n is greater than 10, then neither R² norR³ is a group of the formula (iii). In certain embodiments, when n isgreater than 9, then neither R² nor R³ is a group of the formula (iii).In certain embodiments, when n is greater than 8, then neither R² nor R³is a group of the formula (iii). In certain embodiments, when n isgreater than 7, then neither R² nor R³ is a group of the formula (iii).In certain embodiments, when n is greater than 6, then neither R² nor R³is a group of the formula (iii). In certain embodiments, when n isgreater than 5, then neither R² nor R³ is a group of the formula (iii).In certain embodiments, when n is greater than 4, then neither R² nor R³is a group of the formula (iii). In certain embodiments, when n isgreater than 3, then neither R² nor R³ is a group of the formula (iii).In certain embodiments, when n is greater than 2, then neither R² nor R³is a group of the formula (iii). In certain embodiments, when n isgreater than 1, then neither R² nor R³ is a group of the formula (iii).In certain embodiments, neither R² nor R³ is a group of the formula(iii).

In certain embodiments, wherein n is 0 and Z is 0, one or more of thefollowing compounds are excluded:

and salts thereof; wherein R² is a group of the formula (i), R³ and R⁶are independently hydrogen or a group of formula (i), and Y is O.

As generally defined above, n is 0 or is an integer between 1 and100,000, inclusive. It is thus understood that Formula (I) encompassesamino acids conjugated to a lipid group, as well as linear peptides andlinear polypeptides conjugated to lipid groups.

In certain embodiments, n is 0 or is an integer between 1 and 90,000,inclusive. In certain embodiments, n is 0 or is an integer between 1 and80,000, inclusive. In certain embodiments, n is 0 or is an integerbetween 1 and 70,000, inclusive. In certain embodiments, n is 0 or is aninteger between 1 and 50,000, inclusive. In certain embodiments, n is 0or is an integer between 1 and 40,000, inclusive. In certainembodiments, n is 0 or is an integer between 1 and 30,000, inclusive. Incertain embodiments, n is 0 or is an integer between 1 and 20,000,inclusive. In certain embodiments, n is 0 or is an integer between 1 and10,000, inclusive. In certain embodiments, n is 0 or is an integerbetween 1 and 9,000, inclusive. In certain embodiments, n is 0 or is aninteger between 1 and 8,000, inclusive. In certain embodiments, n is 0or is an integer between 1 and 7,000, inclusive. In certain embodiments,n is 0 or is an integer between 1 and 6,000, inclusive. In certainembodiments, n is 0 or is an integer between 1 and 5,000, inclusive. Incertain embodiments, n is 0 or is an integer between 1 and 4,000,inclusive. In certain embodiments, n is 0 or is an integer between 1 and3,000, inclusive. In certain embodiments, n is 0 or is an integerbetween 1 and 2,000, inclusive. In certain embodiments, n is 0 or is aninteger between 1 and 1,000, inclusive. In certain embodiments, n is 0or is an integer between 1 and 900, inclusive. In certain embodiments, nis 0 or is an integer between 1 and 800, inclusive. In certainembodiments, n is 0 or is an integer between 1 and 700, inclusive. Incertain embodiments, n is 0 or is an integer between 1 and 600,inclusive. In certain embodiments, n is 0 or is an integer between 1 and500, inclusive. In certain embodiments, n is 0 or is an integer between100 and 80,000, inclusive. In certain embodiments, n is 0 or is aninteger between 200 and 80,000, inclusive. In certain embodiments, n is0 or is an integer between 300 and 80,000, inclusive. In certainembodiments, n is 0 or is an integer between 400 and 80,000, inclusive.In certain embodiments, n is 0 or is an integer between 500 and 80,000,inclusive. In certain embodiments, n is 0 or is an integer between 500and 40,000, inclusive. In certain embodiments, n is 0 or is an integerbetween 500 and 30,000, inclusive. In certain embodiments, n is 0 or isan integer between 1 and 400, inclusive. In certain embodiments, n is 0or is an integer between 1 and 300, inclusive. In certain embodiments, nis 0 or is an integer between 1 and 200, inclusive. In certainembodiments, n is 0 or is an integer between 1 and 100, inclusive. Incertain embodiments, n is 0 or is an integer between 1 and 75,inclusive. In certain embodiments, n is 0 or is an integer between 1 and50, inclusive. In certain embodiments, n is 0 or is an integer between 1and 25, inclusive. In certain embodiments, n is 0 or is an integerbetween 1 and 15, inclusive. In certain embodiments, n is 0 or is aninteger between 1 and 10, Inclusive. In certain embodiments, n is 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10.

For example, when n is 0, the compound of Formula (I) is a compound ofthe Formula (I-a):

or salt thereof.

In certain embodiments, when n is 1, the compound of Formula (I) is acompound of the Formula (I-b):

or salt thereof.

In certain embodiments, when n is 2, the compound of Formula (I) is acompound of the Formula (I-c):

or salt thereof.

In certain embodiments, when n is 3, the compound of Formula (I) is acompound of the Formula (I-d):

or salt thereof.

In certain embodiments, when n is 4, the compound of Formula (I) is acompound of the Formula (I-e):

or salt thereof.

As generally defined above, each instance of m is independently 1, 2, or3. In certain embodiments, at least one instance of m is 1. In certainembodiments, each instance of m is 1. In certain embodiments, at leastone instance of m is 2. In certain embodiments, at least one instance ofm is 3.

As generally defined above, each instance of R′ is independentlyhydrogen or optionally substituted alkyl. In certain embodiments, atleast one instance of R′ is hydrogen. In certain embodiments, at leasttwo instances of R′ is hydrogen. In certain embodiments, each instanceof R′ is hydrogen. In certain embodiments, at least one instance of R′is optionally substituted alkyl, e.g., methyl. In certain embodiments,at least two instances of R′ is optionally substituted alkyl, e.g.,methyl. In certain embodiments, one instance of R′ is optionallysubstituted alkyl, and the rest are hydrogen.

As generally defined above, each instance of Z is independently O, S, orNR^(Z), wherein R^(Z) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii), or(iii). In certain embodiments, at least one instance of Z is O. Incertain embodiments, each instance of Z is O. In certain embodiments, atleast one instance of Z is S. In certain embodiments, each instance of Zis S. In certain embodiments, at least one instance of Z is NR^(Z). Incertain embodiments, each instance of Z is NR^(Z). In certainembodiments, each instance of R^(Z) is independently hydrogen or a groupof the formula (i), (ii), or (iii).

As generally defined above, each instance of R¹ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, halogen, —OR^(A1), —N(R^(A1))₂, or—SR^(A1).

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionallysubstituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionallysubstituted C₃₋₄alkyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl,optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl,optionally substituted C₄₋₅alkenyl, or optionally substitutedC₃₋₄alkenyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl,optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl,optionally substituted C₄₋₅alkynyl, or optionally substitutedC₃₋₄alkynyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl,optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted heterocyclyl, e.g., optionally substituted 3-14 memberedheterocyclyl, optionally substituted 3-10 membered heterocyclyl,optionally substituted 5-8 membered heterocyclyl, optionally substituted5-6 membered heterocyclyl, optionally substituted 5 memberedheterocyclyl, or optionally substituted 6 membered heterocyclyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted aryl, e.g., optionally substituted phenyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted heteroaryl, e.g., optionally substituted 5-14 memberedheteroaryl, optionally substituted 5-10 membered heteroaryl, optionallysubstituted 5-6 membered heteroaryl, optionally substituted 5 memberedheteroaryl, or optionally substituted 6 membered heteroaryl.

In any of the above embodiments, the R¹ alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted,for example, with an optionally substituted amino group (e.g., —NR⁶R⁷),an optionally substituted hydroxyl group (e.g., —OR⁶), an optionallysubstituted thiol group (e.g., —SR⁶), or with a group of formula (i),(ii), or (iii), wherein each instance of R⁶ and R⁷ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group whenattached to a nitrogen atom, an oxygen protecting group when attached toan oxygen atom, and a sulfur protecting group when attached to a sulfuratom, or a group of formula (i), (ii), or (iii).

For example, in certain embodiments, at least one instance of R¹ is analkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroarylgroup substituted with an amino group of the formula —N(R⁶)(R⁷). In thatinstance, in certain embodiments, at least one instance of R¹ is a groupof formula:

wherein:

L is an optionally substituted alkylene, optionally substitutedalkenylene, optionally substituted alkynylene, optionally substitutedheteroalkylene, optionally substituted heteroalkenylene, optionallysubstituted heteroalkynylene, optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,or optionally substituted heteroarylene, or combination thereof, and

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, and a nitrogen protecting group;

provided at least one instance of R⁶ and R⁷ is a group of the formula(i), (ii), or (iii):

wherein R′, X, Y, R^(L), and R^(P) are as defined herein.

In certain embodiments, at least two instances of R¹ is a group offormula (iv). In certain embodiments, at least three instances of R¹ isa group of formula (iv). In certain embodiments, at least four instancesof R¹ is a group of formula (iv). In certain embodiments, at least fiveinstances of R¹ is a group of formula (iv). In certain embodiments, eachinstance of R¹ is a group of formula (iv).

In certain embodiments, L is an optionally substituted alkylene; e.g.,optionally substituted C₁₋₅₀alkylene, optionally substitutedC₁₋₄₀alkylene, optionally substituted C₁₋₃₀alkylene, optionallysubstituted C₁₋₂₀alkylene, optionally substituted C₄₋₂₀alkylene,optionally substituted C₆₋₂₀alkylene, optionally substitutedC₈₋₂₀alkylene, optionally substituted C₁₀₋₂₀alkylene, optionallysubstituted C₁₋₆alkylene, optionally substituted C₂₋₆alkylene,optionally substituted C₃₋₆alkylene, optionally substitutedC₄₋₆alkylene, optionally substituted C₄₋₅alkylene, or optionallysubstituted C₃₋₄alkylene.

In certain embodiments, L is an optionally substituted alkenylene, e.g.,optionally substituted C₂₋₅₀alkenylene, optionally substitutedC₂₋₄₀alkenylene, optionally substituted C₂₋₃₀alkenylene, optionallysubstituted C₂₋₂₀alkenylene, optionally substituted C₄₋₂₀alkenylene,optionally substituted C₆₋₂₀alkenylene, optionally substitutedC₈₋₂₀alkenylene, optionally substituted C₁₀₋₂₀alkenylene, optionallysubstituted C₂₋₆alkenylene, optionally substituted C₃₋₆alkenylene,optionally substituted C₄₋₆alkenylene, optionally substitutedC₄₋₅alkenylene, or optionally substituted C₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted alkynylene, e.g.,optionally substituted C₂₋₅₀alkynylene, optionally substitutedC₂₋₄₀alkynylene, optionally substituted C₂₋₃₀alkynylene, optionallysubstituted C₂₋₂₀alkynylene, optionally substituted C₄₋₂₀alkynylene,optionally substituted C₆₋₂₀alkynylene, optionally substitutedC₈₋₂₀alkynylene, optionally substituted C₁₀₋₂₀alkynylene, optionallysubstituted C₂₋₆alkynylene, optionally substituted C₃₋₆alkynylene,optionally substituted C₄₋₆alkynylene, optionally substitutedC₄₋₅alkynylene, or optionally substituted C₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted heteroalkylene;e.g., optionally substituted heteroC₁₋₅₀alkylene, optionally substitutedheteroC₁₋₄₀alkylene, optionally substituted heteroC₁₋₃₀alkylene,optionally substituted heteroC₁₋₂₀alkylene, optionally substitutedheteroC₄₋₂₀alkylene, optionally substituted heteroC₆₋₂₀alkylene,optionally substituted heteroC₈₋₂₀alkylene, optionally substitutedheteroC₁₀₋₂₀alkylene, optionally substituted heteroC₁₋₆alkylene,optionally substituted heteroC₂₋₆alkylene, optionally substitutedheteroC₃₋₆alkylene, optionally substituted heteroC₄₋₆alkylene,optionally substituted heteroC₄₋₅alkylene, or optionally substitutedheteroC₃₋₄alkylene.

In certain embodiments, L is an optionally substituted heteroalkenylene,e.g., optionally substituted heteroC₂₋₅₀alkenylene, optionallysubstituted heteroC₂₋₄₀alkenylene, optionally substitutedheteroC₂₋₃₀alkenylene, optionally substituted heteroC₂₋₂₀alkenylene,optionally substituted heteroC₄₋₂₀alkenylene, optionally substitutedheteroC₆₋₂₀alkenylene, optionally substituted heteroC₈₋₂₀alkenylene,optionally substituted heteroC₁₀₋₂₀alkenylene, optionally substitutedheteroC₂₋₆alkenylene, optionally substituted heteroC₃₋₆alkenylene,optionally substituted heteroC₄₋₆alkenylene, optionally substitutedheteroC₄₋₅alkenylene, or optionally substituted heteroC₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted alkynylene, e.g.,optionally substituted heteroC₂₋₅₀alkynylene, optionally substitutedheteroC₂₋₄₀alkynylene, optionally substituted heteroC₂₋₃₀alkynylene,optionally substituted heteroC₂₋₂₀alkynylene, optionally substitutedheteroC₄₋₂₀alkynylene, optionally substituted heteroC₆₋₂₀alkynylene,optionally substituted heteroC₈₋₂₀alkynylene, optionally substitutedheteroC₁₀₋₂₀alkynylene, optionally substituted heteroC₂₋₆alkynylene,optionally substituted C₃₋₆alkynylene, optionally substitutedheteroC₄₋₆alkynylene, optionally substituted heteroC₄₋₅alkynylene, oroptionally substituted heteroC₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted carbocyclylene,e.g., optionally substituted C₃₋₁₀ carbocyclylene, optionallysubstituted C₅₋₈ carbocyclylene, optionally substituted C₅₋₆carbocyclylene, optionally substituted C₅ carbocyclylene, or optionallysubstituted C₆ carbocyclylene.

In certain embodiments, L is an optionally substituted heterocyclylene,e.g., optionally substituted 3-14 membered heterocyclylene, optionallysubstituted 3-10 membered heterocyclylene, optionally substituted 5-8membered heterocyclylene, optionally substituted 5-6 memberedheterocyclylene, optionally substituted 5 membered heterocyclylene, oroptionally substituted 6 membered heterocyclylene.

In certain embodiments, L is an optionally substituted arylene, e.g.,optionally substituted phenylene.

In certain embodiments, L is an optionally substituted heteroarylene,e.g., optionally substituted 5-14 membered heteroarylene, optionallysubstituted 5-10 membered heteroarylene, optionally substituted 5-6membered heteroarylene, optionally substituted 5 membered heteroarylene,or optionally substituted 6 membered heteroarylene.

For example, in certain embodiments, wherein L is an optionallysubstituted alkylene group, the group of formula (iv) is a group of theformula:

wherein q is an integer between 1 and 50, inclusive.

In certain embodiments, q is an integer between 1 and 40, inclusive. Incertain embodiments, q is an integer between 1 and 30, inclusive. Incertain embodiments, q is an integer between 1 and 20, inclusive. Incertain embodiments, q is an integer between 4 and 20, inclusive. Incertain embodiments, q is an integer between 6 and 20, inclusive. Incertain embodiments, q is an integer between 8 and 20, inclusive. Incertain embodiments, q is 1. In certain embodiments, q is 2. In certainembodiments, q is 3. In certain embodiments, q is 4. In certainembodiments, q is 5. In certain embodiments, q is 6. In certainembodiments, q is 7. In certain embodiments, q is 8. In certainembodiments, q is 9. In certain embodiments, q is 10.

In certain embodiments, both R⁶ and R⁷ are hydrogen. In certainembodiments, R⁶ is hydrogen and R⁷ is a group of the formula (i), (ii),or (iii). In certain embodiments, R⁶ is hydrogen and R⁷ is a group ofthe formula (i). In certain embodiments, R⁶ is hydrogen and R⁷ is agroup of the formula (ii). In certain embodiments, R⁶ is hydrogen and R⁷is a group of the formula (iii). In certain embodiments, both R⁶ and R⁷are independently a group of the formula (i), (ii), or (iii). In certainembodiments, both R⁶ and R⁷ are independently a group of the formula(i). In certain embodiments, both R⁶ and R⁷ are independently a group ofthe formula (ii). In certain embodiments, both R⁶ and R⁷ areindependently a group of the formula (iii). In certain embodiments, bothR⁶ and R⁷ are the same group, selected from a group of the formula (i),(ii), or (iii).

It is understood that R¹ encompasses amino acid side chains such asexemplified in Table 1 of the Examples. In certain embodiments, R¹ is agroup selected from any one of the amino acid side chain groups listedtherein.

In certain embodiments, each instance of R¹ is the same. In certainembodiments, at least one R¹ group is different. In certain embodiments,each R¹ group is different.

As generally defined above, R² is a group of the formula (i), (ii), or(iii):

wherein R′, X, Y, R^(L), and R^(P) are as defined herein.

In certain embodiments, R² is a group of the formula (i). In certainembodiments, R² is a group of the formula (ii). In certain embodiments,R² is a group of the formula (iii).

As generally defined above, R³ is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii), or(iii); optionally wherein R³ and an R¹ group are joined to form anoptionally substituted 5-6 membered heterocyclic ring;

In certain embodiments, R³ is hydrogen. In certain embodiments, R³ isoptionally substituted alkyl; e.g., optionally substituted C₁₋₆alkyl,optionally substituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl,optionally substituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, oroptionally substituted C₃₋₄alkyl.

In certain embodiments, R³ is optionally substituted alkenyl, e.g.,optionally substituted C₂₋₆alkenyl, optionally substituted C₃₋₆alkenyl,optionally substituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl,or optionally substituted C₃₋₄alkenyl.

In certain embodiments, R³ is optionally substituted alkynyl, e.g.,optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl,optionally substituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl,or optionally substituted C₃₋₄alkynyl.

In certain embodiments, R³ is optionally substituted carbocyclyl, e.g.,optionally substituted C₃₋₁₀ carbocyclyl, optionally substituted C₅₋₈carbocyclyl, optionally substituted C₅₋₆ carbocyclyl, optionallysubstituted C₅ carbocyclyl, or optionally substituted C₆ carbocyclyl.

In certain embodiments, R³ is optionally substituted heterocyclyl, e.g.,optionally substituted 3-14 membered heterocyclyl, optionallysubstituted 3-10 membered heterocyclyl, optionally substituted 5-8membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl,optionally substituted 5 membered heterocyclyl, or optionallysubstituted 6 membered heterocyclyl.

In certain embodiments, R³ is optionally substituted aryl, e.g.,optionally substituted phenyl.

In certain embodiments, R³ is optionally substituted heteroaryl, e.g.,optionally substituted 5-14 membered heteroaryl, optionally substituted5-10 membered heteroaryl, optionally substituted 5-6 memberedheteroaryl, optionally substituted 5 membered heteroaryl, or optionallysubstituted 6 membered heteroaryl.

In certain embodiments, R³ is a nitrogen protecting group.

In certain embodiments, R³ is group of the formula (i), (ii), or (iii).In certain embodiments, R³ is group of the formula (i). In certainembodiments, R³ is group of the formula (ii). In certain embodiments, R³is group of the formula (iii).

In certain embodiments, R³ and an adjacent R¹ group are joined to forman optionally substituted 5-6 membered heterocyclic ring, e.g., a5-membered heterocyclic ring, e.g., an optionally substitutedpyrrolidinyl ring.

In certain embodiments, R³ is hydrogen and R² is a group of the formula(i), (ii), or (iii). In certain embodiments, R³ is hydrogen and R² is agroup of the formula (i). In certain embodiments, R³ is hydrogen and R²is a group of the formula (ii). In certain embodiments, R³ is hydrogenand R² is a group of the formula (iii). In certain embodiments, both R²and R³ are independently a group of the formula (i), (ii), or (iii). Incertain embodiments, both R² and R³ are independently a group of theformula (i). In certain embodiments, both R² and R³ are independently agroup of the formula (ii). In certain embodiments, both R² and R³ areindependently a group of the formula (iii). In certain embodiments, bothR² and R³ are the same group, selected from a group of the formula (i),(ii), or (iii).

As generally defined above, R⁴ is —OR^(A4), —N(R^(A4))₂, or —SR^(A4);wherein each occurrence of R^(A4) is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, an oxygen protecting group when attached to anoxygen atom, a sulfur protecting group when attached to an sulfur atom,a nitrogen protecting group when attached to a nitrogen atom, or twoR^(A4) groups are joined to form an optionally substituted heterocyclicor optionally substituted heteroaryl ring.

In certain embodiments, R⁴ is —OR^(A4), wherein R^(A4) is hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, or an oxygen protecting group. In certainembodiments, R^(A4) is hydrogen or optionally substituted alkyl. Incertain embodiments, R^(A4) is hydrogen.

In certain embodiments, R⁴ is —N(R^(A4))₂, wherein each occurrence ofR^(A4) is independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group when attached to a nitrogen atom, or twoR^(A4) groups are joined to form an optionally substituted heterocyclicor optionally substituted heteroaryl ring. In certain embodiments, atleast one instance of R^(A4) is hydrogen or optionally substitutedalkyl. In certain embodiments, at least one instance of R^(A4) ishydrogen.

In certain embodiments, R⁴ is —SR^(A4), wherein R^(A4) is hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, or sulfur protecting group. In certainembodiments, R^(A4) is hydrogen or optionally substituted alkyl. Incertain embodiments, R^(A4) is hydrogen.

As generally defined above, R⁵ is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group. In certain embodiments, at least one instanceof R⁵ is hydrogen. In certain embodiments, each instance of R⁵ ishydrogen.

Various combinations of the above embodiments of Formula (I) arecontemplated herein.

For example, in certain embodiments, wherein each instance of m is 1 andeach instance of Z is 0, the compound of Formula (I) is a compound ofFormula (I-f):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, R² is a group of formula (i). Incertain embodiments, R² is a group of formula (ii). In certainembodiments, R² is a group of formula (iii). In certain embodiments, R³is a group of formula (i). In certain embodiments, R³ is a group offormula (ii). In certain embodiments, R³ is a group of formula (iii). Incertain embodiments, R⁴ is —OR^(A4). In certain embodiments, R⁵ ishydrogen. In certain embodiments, n is 0. In certain embodiments, nis 1. In certain embodiments, n is 2. In certain embodiments, n is 3. Incertain embodiments, n is 4. In certain embodiments, n is 5.

For example, in certain embodiments of Formula (I-f), wherein eachinstance of R¹ is a group of the formula (iv), provided is a compound ofFormula (I-f1):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii).

In certain embodiments of Formula (I-f), wherein R² is a group offormula (i), the compound is of Formula (I-f2):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, R³ is a group of formula (i). Incertain embodiments, R³ is a group of formula (ii). In certainembodiments, R³ is a group of formula (iii). In certain embodiments, R⁴is —OR^(A4). In certain embodiments, R⁵ is hydrogen. In certainembodiments, n is 0. In certain embodiments, n is 1. In certainembodiments, n is 2. In certain embodiments, n is 3. In certainembodiments, n is 4. In certain embodiments, n is 5.

For example, in certain embodiments of Formula (I-f2), wherein eachinstance of R¹ is a group of the formula (iv), provided is a compound ofFormula (I-f3):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii).

In certain embodiments of Formula (I-f), wherein R² and R³ are eachindependently a group of formula (i), the compound is of Formula (I-f4):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, R⁴ is —OR^(A4). In certainembodiments, R⁵ is hydrogen. In certain embodiments, n is 0. In certainembodiments, n is 1. In certain embodiments, n is 2. In certainembodiments, n is 3. In certain embodiments, n is 4. In certainembodiments, n is 5.

For example, in certain embodiments of Formula (I-f4), wherein eachinstance of R¹ is a group of the formula (iv), provided is a compound ofFormula (I-f5):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii).

In certain embodiments of Formula (I-f), wherein R² is a group offormula (ii), the compound is of Formula (I-f6):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, R³ is a group of formula (i). Incertain embodiments, R³ is a group of formula (ii). In certainembodiments, R³ is a group of formula (iii). In certain embodiments, R⁴is —OR^(A4). In certain embodiments, R⁵ is hydrogen. In certainembodiments, n is 0. In certain embodiments, n is 1. In certainembodiments, n is 2. In certain embodiments, n is 3. In certainembodiments, n is 4. In certain embodiments, n is 5.

For example, in certain embodiments of Formula (I-f6), wherein eachinstance of R¹ is a group of the formula (iv), provided is a compound ofFormula (I-f7):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii).

In certain embodiments of Formula (I-f), wherein R² and R³ areindependently a group of formula (ii), the compound is of Formula(I-f8):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, R⁴ is —OR^(A4). In certainembodiments, R⁵ is hydrogen. In certain embodiments, n is 0. In certainembodiments, n is 1. In certain embodiments, n is 2. In certainembodiments, n is 3. In certain embodiments, n is 4. In certainembodiments, n is 5.

For example, in certain embodiments of Formula (I-f8), wherein eachinstance of R¹ is a group of the formula (iv), provided is a compound ofFormula (I-f9):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii).

In certain embodiments of Formula (I-f), wherein R² is a group offormula (iii), the compound is of Formula (I-f10):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, R³ is a group of formula (i). Incertain embodiments, R³ is a group of formula (ii). In certainembodiments, R³ is a group of formula (iii). In certain embodiments, R⁴is —OR^(A4). In certain embodiments, R⁵ is hydrogen. In certainembodiments, n is 0. In certain embodiments, n is 1. In certainembodiments, n is 2. In certain embodiments, n is 3. In certainembodiments, n is 4. In certain embodiments, n is 5.

For example, in certain embodiments of Formula (I-f10), wherein eachinstance of R¹ is a group of the formula (iv), provided is a compound ofFormula (I-f11):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii).

In certain embodiments of Formula (I-f), wherein R² and R³ areindependently a group of formula (iii), the compound is of Formula(I-f12):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, R⁴ is —OR^(A4). In certainembodiments, R⁵ is hydrogen. In certain embodiments, n is 0. In certainembodiments, n is 1. In certain embodiments, n is 2. In certainembodiments, n is 3. In certain embodiments, n is 4. In certainembodiments, n is 5.

For example, in certain embodiments of Formula (I-f12), wherein eachinstance of R¹ is a group of the formula (iv), provided is a compound ofFormula (I-f13):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii).

Compounds of Formula (II)

Compounds of Formula (II) may be prepared via internal cyclization ofthe addition product of a primary or secondary amine or amide of anamino acid, peptide, or polypeptide, and an epoxide, thiirane, oraziridine of formula (i-x) (Scheme 2).

Compounds of Formula (II) may encompass additional sites of conjugation,e.g., the secondary amino group, appended to a group attached to thesecondary amino group, an amino substituent, and/or an imino nitrogen,to a group of formula (i), (ii), or (iii):

Thus, in a second aspect, provided is a compound of Formula (II):

or salt thereof;wherein:

each instance of R′ is independently hydrogen or optionally substitutedalkyl;

each instance of R¹ is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, halogen,—OR^(A1), —N(R^(A1))₂, or —SR^(A1); wherein each occurrence of R^(A1) isindependently hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, an oxygenprotecting group when attached to an oxygen atom, a sulfur protectinggroup when attached to an sulfur atom, a nitrogen protecting group whenattached to a nitrogen atom, or two R^(A1) groups are joined to form anoptionally substituted heterocyclic or optionally substituted heteroarylring;

R⁸ is hydrogen, a group of the formula (i), (ii), or (iii), or a groupof the formula (v):

wherein Z, R², R³, R⁵, m, and n are as defined for Formula (I);

or R⁸ and an R¹ group are joined to form an optionally substituted 5-6membered heterocyclic ring;

each instance of W is independently O, S, or NR^(W), wherein R^(W) ishydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group, or agroup of the formula (i), (ii), or (iii); and

each instance of Y is independently O, S, or NR^(Y), wherein R^(Y) ishydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, or a nitrogen protecting group;Formulae (i), (ii), and (iii) are:

wherein:

X is O, S, NR^(X), wherein R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

Y is O, S, NR^(Y), wherein R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

R^(P) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached to asulfur atom, or a nitrogen protecting group when attached to a nitrogenatom; and

R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substitutedC₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionallysubstituted heteroC₁₋₅₀alkyl, optionally substituted heteroC₂₋₅₀alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.

In certain embodiments, wherein Y is O and W is O, the followingcompounds are specifically excluded:

wherein R⁸ and R⁶ are independently hydrogen or a group of formula (i),and salts thereof.

In certain embodiments, at least one instance of R^(W), R², R³, R⁶, R⁷,or R⁸ is a group of the formula (i), (ii), or (iii).

As generally defined above, each instance of R′ is independentlyhydrogen or optionally substituted alkyl. In certain embodiments, atleast one instance of R′ is hydrogen. In certain embodiments, at leasttwo instances of R′ is hydrogen. In certain embodiments, each instanceof R′ is hydrogen. In certain embodiments, at least one instance of R′is optionally substituted alkyl, e.g., methyl. In certain embodiments,at least two instances of R′ is optionally substituted alkyl, e.g.,methyl. In certain embodiments, one instance of R′ is optionallysubstituted alkyl, and the rest are hydrogen.

As generally defined above, each instance of R¹ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, halogen, —OR^(A1), —N(R^(A1))₂, or—SR^(A1).

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionallysubstituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionallysubstituted C₃₋₄alkyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl,optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl,optionally substituted C₄₋₅alkenyl, or optionally substitutedC₃₋₄alkenyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl,optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl,optionally substituted C₄₋₅alkynyl, or optionally substitutedC₃₋₄alkynyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl,optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted heterocyclyl, e.g., optionally substituted 3-14 memberedheterocyclyl, optionally substituted 3-10 membered heterocyclyl,optionally substituted 5-8 membered heterocyclyl, optionally substituted5-6 membered heterocyclyl, optionally substituted 5 memberedheterocyclyl, or optionally substituted 6 membered heterocyclyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted aryl, e.g., optionally substituted phenyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted heteroaryl, e.g., optionally substituted 5-14 memberedheteroaryl, optionally substituted 5-10 membered heteroaryl, optionallysubstituted 5-6 membered heteroaryl, optionally substituted 5 memberedheteroaryl, or optionally substituted 6 membered heteroaryl.

In any of the above embodiments, the R¹ alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted,for example, with an optionally substituted amino group (e.g., —NR⁶R⁷),an optionally substituted hydroxyl group (e.g., —OR⁶), an optionallysubstituted thiol group (e.g., —SR⁶), or with a group of formula (i),(ii), or (iii), wherein each instance of R⁶ and R⁷ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group whenattached to a nitrogen atom, an oxygen protecting group when attached toan oxygen atom, and a sulfur protecting group when attached to a sulfuratom, or a group of formula (i), (ii), or (iii).

For example, in certain embodiments, at least one instance of R¹ is analkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroarylgroup substituted with an amino group of the formula —N(R⁶)(R⁷). In thatinstance, in certain embodiments, at least one instance of R¹ is a groupof formula:

wherein:

L is an optionally substituted alkylene, optionally substitutedalkenylene, optionally substituted alkynylene, optionally substitutedheteroalkylene, optionally substituted heteroalkenylene, optionallysubstituted heteroalkynylene, optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,or optionally substituted heteroarylene, or combination thereof, and

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, and a nitrogen protecting group;

provided at least one instance of R⁶ and R⁷ is a group of the formula(i), (ii), or (iii):

wherein R′, X, Y, R^(L), and R^(P) are as defined herein.

In certain embodiments, at least two instances of R¹ is a group offormula (iv). In certain embodiments, at least three instances of R¹ isa group of formula (iv). In certain embodiments, at least four instancesof R¹ is a group of formula (iv). In certain embodiments, at least fiveinstances of R¹ is a group of formula (iv). In certain embodiments, eachinstance of R¹ is a group of formula (iv).

In certain embodiments, R¹ alpha to the group —C(═W)—Y— is a group offormula (iv).

In certain embodiments, at least one instance of R¹ provided in group R⁸is a group of formula (iv). In certain embodiments, at least twoinstances of R¹ provided in group R⁸ is a group of formula (iv). Incertain embodiments, at least three instances of R¹ provided in group R⁸is a group of formula (iv). In certain embodiments, at least fourinstances of R¹ provided in group R⁸ is a group of formula (iv). Incertain embodiments, at least five instances of R¹ provided in group R⁸is a group of formula (iv). In certain embodiments, each instance of R¹provided in group R⁸ is a group of formula (iv).

In certain embodiments, L is an optionally substituted alkylene; e.g.,optionally substituted C₁₋₅₀alkylene, optionally substitutedC₁₋₄₀alkylene, optionally substituted C₁₋₃₀alkylene, optionallysubstituted C₁₋₂₀alkylene, optionally substituted C₄₋₂₀alkylene,optionally substituted C₆₋₂₀alkylene, optionally substitutedC₈₋₂₀alkylene, optionally substituted C₁₀₋₂₀alkylene, optionallysubstituted C₁₋₆alkylene, optionally substituted C₂₋₆alkylene,optionally substituted C₃₋₆alkylene, optionally substitutedC₄₋₆alkylene, optionally substituted C₄₋₅alkylene, or optionallysubstituted C₃₋₄alkylene.

In certain embodiments, L is an optionally substituted alkenylene, e.g.,optionally substituted C₂₋₅₀alkenylene, optionally substitutedC₂₋₄₀alkenylene, optionally substituted C₂₋₃₀alkenylene, optionallysubstituted C₂₋₂₀alkenylene, optionally substituted C₄₋₂₀alkenylene,optionally substituted C₆₋₂₀alkenylene, optionally substitutedC₈₋₂₀alkenylene, optionally substituted C₁₀₋₂₀alkenylene, optionallysubstituted C₂₋₆alkenylene, optionally substituted C₃₋₆alkenylene,optionally substituted C₄₋₆alkenylene, optionally substitutedC₄₋₅alkenylene, or optionally substituted C₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted alkynylene, e.g.,optionally substituted C₂₋₅₀alkynylene, optionally substitutedC₂₋₄₀alkynylene, optionally substituted C₂₋₃₀alkynylene, optionallysubstituted C₂₋₂₀alkynylene, optionally substituted C₄₋₂₀alkynylene,optionally substituted C₆₋₂₀alkynylene, optionally substitutedC₈₋₂₀alkynylene, optionally substituted C₁₀₋₂₀alkynylene, optionallysubstituted C₂₋₆alkynylene, optionally substituted C₃₋₆alkynylene,optionally substituted C₄₋₆alkynylene, optionally substitutedC₄₋₅alkynylene, or optionally substituted C₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted heteroalkylene;e.g., optionally substituted heteroC₁₋₅₀alkylene, optionally substitutedheteroC₁₋₄₀alkylene, optionally substituted heteroC₁₋₃₀alkylene,optionally substituted heteroC₁₋₂₀alkylene, optionally substitutedheteroC₄₋₂₀alkylene, optionally substituted heteroC₆₋₂₀alkylene,optionally substituted heteroC₈₋₂₀alkylene, optionally substitutedheteroC₁₀₋₂₀alkylene, optionally substituted heteroC₁₋₆alkylene,optionally substituted heteroC₂₋₆alkylene, optionally substitutedheteroC₃₋₆alkylene, optionally substituted heteroC₄₋₆alkylene,optionally substituted heteroC₄₋₅alkylene, or optionally substitutedheteroC₃₋₄alkylene.

In certain embodiments, L is an optionally substituted heteroalkenylene,e.g., optionally substituted heteroC₂₋₅₀alkenylene, optionallysubstituted heteroC₂₋₄₀alkenylene, optionally substitutedheteroC₂₋₃₀alkenylene, optionally substituted heteroC₂₋₂₀alkenylene,optionally substituted heteroC₄₋₂₀alkenylene, optionally substitutedheteroC₆₋₂₀alkenylene, optionally substituted heteroC₈₋₂₀alkenylene,optionally substituted heteroC₁₀₋₂₀alkenylene, optionally substitutedheteroC₂₋₆alkenylene, optionally substituted heteroC₃₋₆alkenylene,optionally substituted heteroC₄₋₆alkenylene, optionally substitutedheteroC₄₋₅alkenylene, or optionally substituted heteroC₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted alkynylene, e.g.,optionally substituted heteroC₂₋₅₀alkynylene, optionally substitutedheteroC₂₋₄₀alkynylene, optionally substituted heteroC₂₋₃₀alkynylene,optionally substituted heteroC₂₋₂₀alkynylene, optionally substitutedheteroC₄₋₂₀alkynylene, optionally substituted heteroC₆₋₂₀alkynylene,optionally substituted heteroC₈₋₂₀alkynylene, optionally substitutedheteroC₁₀₋₂₀alkynylene, optionally substituted heteroC₂₋₆alkynylene,optionally substituted C₃₋₆alkynylene, optionally substitutedheteroC₄₋₆alkynylene, optionally substituted heteroC₄₋₅alkynylene, oroptionally substituted heteroC₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted carbocyclylene,e.g., optionally substituted C₃₋₁₀ carbocyclylene, optionallysubstituted C₅₋₈ carbocyclylene, optionally substituted C₅₋₆carbocyclylene, optionally substituted C₅ carbocyclylene, or optionallysubstituted C₆ carbocyclylene.

In certain embodiments, L is an optionally substituted heterocyclylene,e.g., optionally substituted 3-14 membered heterocyclylene, optionallysubstituted 3-10 membered heterocyclylene, optionally substituted 5-8membered heterocyclylene, optionally substituted 5-6 memberedheterocyclylene, optionally substituted 5 membered heterocyclylene, oroptionally substituted 6 membered heterocyclylene.

In certain embodiments, L is an optionally substituted arylene, e.g.,optionally substituted phenylene.

In certain embodiments, L is an optionally substituted heteroarylene,e.g., optionally substituted 5-14 membered heteroarylene, optionallysubstituted 5-10 membered heteroarylene, optionally substituted 5-6membered heteroarylene, optionally substituted 5 membered heteroarylene,or optionally substituted 6 membered heteroarylene.

For example, in certain embodiments, wherein L is an optionallysubstituted alkylene group, the group of formula (iv) is a group of theformula:

wherein q is an integer between 1 and 50, inclusive.

In certain embodiments, q is an integer between 1 and 40, inclusive. Incertain embodiments, q is an integer between 1 and 30, inclusive. Incertain embodiments, q is an integer between 1 and 20, inclusive. Incertain embodiments, q is an integer between 4 and 20, inclusive. Incertain embodiments, q is an integer between 6 and 20, inclusive. Incertain embodiments, q is an integer between 8 and 20, inclusive. Incertain embodiments, q is 1. In certain embodiments, q is 2. In certainembodiments, q is 3. In certain embodiments, q is 4. In certainembodiments, q is 5. In certain embodiments, q is 6. In certainembodiments, q is 7. In certain embodiments, q is 8. In certainembodiments, q is 9. In certain embodiments, q is 10.

In certain embodiments, both R⁶ and R⁷ are hydrogen. In certainembodiments, R⁶ is hydrogen and R⁷ is a group of the formula (i), (ii),or (iii). In certain embodiments, R⁶ is hydrogen and R⁷ is a group ofthe formula (i). In certain embodiments, R⁶ is hydrogen and R⁷ is agroup of the formula (ii). In certain embodiments, R⁶ is hydrogen and R⁷is a group of the formula (iii). In certain embodiments, both R⁶ and R⁷are independently a group of the formula (i), (ii), or (iii). In certainembodiments, both R⁶ and R⁷ are independently a group of the formula(i). In certain embodiments, both R⁶ and R⁷ are independently a group ofthe formula (ii). In certain embodiments, both R⁶ and R⁷ areindependently a group of the formula (iii). In certain embodiments, bothR⁶ and R⁷ are the same group, selected from a group of the formula (i),(ii), or (iii).

It is understood that R¹ encompasses amino acid side chains such asexemplified in Table 1 of the Examples. In certain embodiments, R¹ is agroup selected from any one of the amino acid side chain groups listedtherein.

In certain embodiments, each instance of R¹ is the same. In certainembodiments, at least one R¹ group is different. In certain embodiments,each R¹ group is different.

As generally defined above, each instance of W is independently O, S, orNR^(W), wherein R^(W) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii), or(iii). In certain embodiments, W is O. In certain embodiments, W is S.In certain embodiments, W is NR^(W). In certain embodiments, R^(W) ishydrogen or a group of the formula (i), (ii), or (iii).

As generally defined above, each instance of Y is independently O, S, orNR^(Y), wherein R^(Y) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group. In certain embodiments, Y is O. In certainembodiments, each instance of Y is S. In certain embodiments, Y isNR^(Y). In certain embodiments, R^(Y) is hydrogen or a nitrogenprotecting group.

In certain embodiments, W is O and Y is O. In certain embodiments, W isO and Y is S. In certain embodiments, W is O and Y is NR^(Y). In certainembodiments, W is S and Y is O. In certain embodiments, W is S and Y isS. In certain embodiments, W is S and Y is NR^(Y). In certainembodiments, W is NR^(W) and Y is O. In certain embodiments, W is NR^(W)and Y is S. In certain embodiments, W is NR^(W) and Y is NR^(Y).

As generally defined above, R⁸ is hydrogen, a group of the formula (i),(ii), or (iii), or a group of the formula (v):

wherein R², R³, R⁵, Z, m, and n are as defined in Formula (I), providedat least one instance of R^(W), R², R³, R⁸, R⁶, R⁷, or R⁸ is a group ofthe formula (i), (ii), or (iii).

In certain embodiments, R⁸ is hydrogen.

In certain embodiments, R⁸ is a group of the formula (i), (ii), or(iii). In certain embodiments, R⁸ is a group of the formula (i). Incertain embodiments, R⁸ is a group of the formula (ii). In certainembodiments, R⁸ is a group of the formula (iii).

In certain embodiments, R⁸ is a group of the formula (v). In certainembodiments, R⁸ is a group of the formula (v) and R² is a group of theformula (i), (ii), or (iii). In certain embodiments, R⁸ is a group ofthe formula (v) and R³ is a group of the formula (i), (ii), or (iii).

In certain embodiments, at least one R¹ is a group of formula (iv) andR⁶ is a group of the formula (i), (ii), or (iii). In certainembodiments, at least one R¹ is a group of formula (iv) and R⁷ is agroup of the formula (i), (ii), or (iii). In certain embodiments, atleast one R¹ is a group of formula (iv), and both R⁶ and R⁷ areindependently groups of the formula (i), (ii), or (iii).

Alternatively, in certain embodiments, R⁸ and the adjacent R¹ group arejoined to form an optionally substituted 5-6 membered heterocyclic ring,e.g., a 5-membered heterocyclic ring, e.g., an optionally substitutedpyrrolidinyl ring.

Various combinations of the above embodiments of Formula (II) arecontemplated herein.

For example, in certain embodiments, wherein each instance of R′ ishydrogen, W is O and Y is O, the compound of Formula (II) is a compoundof Formula (II-a):

or salt thereof. In certain embodiments, R⁸ is a group of the formula(i), (ii), or (iii). In certain embodiments, R⁸ is a group of theformula (v) and R² is a group of the formula (i), (ii), or (iii). Incertain embodiments, R⁸ is a group of the formula (v) and R³ is a groupof the formula (i), (ii), or (iii). In certain embodiments, at least oneR¹ is a group of formula (iv). In certain embodiments, R¹ is a group offormula (iv) and R⁶ is a group of the formula (i), (ii), or (iii). Incertain embodiments, R¹ is a group of formula (iv) and R⁷ is a group ofthe formula (i), (ii), or (iii). In certain embodiments, both R⁶ and R⁷are independently groups of the formula (i), (ii), or (iii).

In certain embodiments of Formula (II-a), wherein R¹ alpha to the group—C(═O)—O— is a group of formula (iv), provided is a compound of Formula(II-b):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii). Incertain embodiments, both R⁶ and R⁷ are independently groups of theformula (i), (ii), or (iii).

In certain embodiments of Formula (II-a), wherein R⁸ is a group offormula (v), provided is a compound of Formula (II-c):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, R² is a group of formula (i). Incertain embodiments, R² is a group of formula (ii). In certainembodiments, R² is a group of formula (iii). In certain embodiments, R³is a group of formula (i). In certain embodiments, R³ is a group offormula (ii). In certain embodiments, R³ is a group of formula (iii). Incertain embodiments, R⁵ is hydrogen. In certain embodiments, Z is O. Incertain embodiments, n is 0. In certain embodiments, n is 1. In certainembodiments, n is 2. In certain embodiments, n is 3. In certainembodiments, n is 4. In certain embodiments, n is 5. In certainembodiments, m is 1.

In certain embodiments of Formula (II-c), wherein R¹ alpha to the group—C(═O)—O— is a group of formula (iv), provided is a compound of Formula(II-c1):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii). Incertain embodiments, both R⁶ and R⁷ are independently groups of theformula (i), (ii), or (iii).

In certain embodiments of Formula (II-c), wherein each instance of R¹provided in group R⁸ is a group of formula (iv), provided is a compoundof Formula (II-c2):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii). Incertain embodiments, both R⁶ and R⁷ are independently groups of theformula (i), (ii), or (iii).

In certain embodiments of Formula (II-c), wherein each instance of R¹ isa group of formula (iv), provided is a compound of Formula (II-c3):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii). Incertain embodiments, both R⁶ and R⁷ are independently groups of theformula (i), (ii), or (iii).

In certain embodiments of Formula (II-a), wherein R⁸ is a group offormula (i), provided is a compound of Formula (II-d):

or salt thereof. In certain embodiments, R¹ is hydrogen. In certainembodiments, R¹ is a group of formula (iv). In certain embodiments, R¹is a group of formula (iv) and R⁶ is a group of the formula (i), (ii),or (iii). In certain embodiments, R¹ is a group of formula (iv) and bothR⁶ and R⁷ are independently groups of the formula (i), (ii), or (iii).

In certain embodiments of Formula (II-d), wherein R¹ alpha to the group—C(═O)—O— is a group of formula (iv), provided is a compound of Formula(II-dl):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii). Incertain embodiments, both R⁶ and R⁷ are independently groups of theformula (i), (ii), or (iii).

In certain embodiments of Formula (II-a), wherein R⁸ is a group offormula (ii), provided is a compound of Formula (II-e):

or salt thereof. In certain embodiments, R¹ is hydrogen. In certainembodiments, R¹ is a group of formula (iv). In certain embodiments, R¹is a group of formula (iv) and R⁶ is a group of the formula (i), (ii),or (iii). In certain embodiments, R¹ is a group of formula (iv) and bothR⁶ and R⁷ are independently groups of the formula (i), (ii), or (iii).

In certain embodiments of Formula (II-e), wherein R¹ alpha to the group—C(═O)—O— is a group of formula (iv), provided is a compound of Formula(II-e1):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii). Incertain embodiments, both R⁶ and R⁷ are independently groups of theformula (i), (ii), or (iii).

In certain embodiments of Formula (II-a), wherein R⁸ is a group offormula (iii), provided is a compound of Formula (II-f):

or salt thereof. In certain embodiments, R¹ is hydrogen. In certainembodiments, R¹ is a group of formula (iv). In certain embodiments, R¹is a group of formula (iv) and R⁶ is a group of the formula (i), (ii),or (iii). In certain embodiments, R¹ is a group of formula (iv) and bothR⁶ and R⁷ are independently groups of the formula (i), (ii), or (iii).

In certain embodiments of Formula (II-f), wherein R¹ alpha to the group—C(═O)—O— is a group of formula (iv), provided is a compound of Formula(II-f1):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii). Incertain embodiments, both R⁶ and R⁷ are independently groups of theformula (i), (ii), or (iii).

In certain embodiments of Formula (II-a), wherein R¹ and R⁸ are joinedto form an optionally substituted 5-6 membered heterocyclic ring,provided is a compound of Formula (II-g):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene.

Compounds of Formula (III)

Compounds of Formula (III) are the cyclic condensation product of thesame or different two, three, four, five, six, seven, eight, nine, orten amino acids, and which further comprise one or more sites ofconjugation attached thereto, e.g., to an internal amide nitrogen, to anamino substituent, and/or to an imino nitrogen, of a group of formula(i), (iii), or (iii). Such groups may be conjugated before cyclization,i.e., to the amino acid precursors of the cyclization product, or aftercyclization.

Thus, in a third aspect, provided is a compound of Formula (III):

or salt thereof;wherein:

p is an integer of between 1 and 9, inclusive;

each instance of Q is independently O, S, or NR^(Q), wherein R^(Q) ishydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group, or agroup of the formula (i), (ii), (iii);

each instance of R¹ is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, halogen,—OR^(A1), —N(R^(A1))₂, or —SR^(A1); wherein each occurrence of R^(A1) isindependently hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, an oxygenprotecting group when attached to an oxygen atom, a sulfur protectinggroup when attached to an sulfur atom, a nitrogen protecting group whenattached to a nitrogen atom, or two R^(A1) groups are joined to form anoptionally substituted heterocyclic or optionally substituted heteroarylring; and

each instance of R² is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii), or(iii); and

Formulae (i), (ii), and (iii) are:

wherein:

each instance of R′ is independently hydrogen or optionally substitutedalkyl;

X is O, S, NR^(X), wherein R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

Y is O, S, NR^(Y), wherein R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

R^(P) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached to asulfur atom, or a nitrogen protecting group when attached to a nitrogenatom; and

R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substitutedC₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionallysubstituted heteroC₁₋₅₀alkyl, optionally substituted heteroC₂₋₅₀alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer;

provided that at least one instance of R^(Q), R², R⁶, or R⁷ is a groupof the formula (i), (ii), or (iii).

As generally defined above, p is an integer of between 1 and 9,inclusive. In certain embodiments, p is 1. In certain embodiments, p is2. In certain embodiments, p is 3. In certain embodiments, p is 4. Incertain embodiments, p is 5. In certain embodiments, p is 6. In certainembodiments, p is 7. In certain embodiments, p is 8. In certainembodiments, p is 9.

For example, in certain embodiments, wherein p is 1, the compound ofFormula (III) is a compound of Formula (III-a):

or salt thereof.

In certain embodiments, wherein p is 2, the compound of Formula (III) isa compound of Formula (III-b):

or salt thereof.

In certain embodiments, wherein p is 3, the compound of Formula (III) isa compound of Formula (III-c):

or salt thereof.

In certain embodiments, wherein p is 4, the compound of Formula (III) isa compound of Formula (III-d):

or salt thereof.

In certain embodiments, wherein p is 5, the compound of Formula (III) isa compound of Formula (III-e):

or salt thereof.

In certain embodiments, wherein p is 6, the compound of Formula (III) isa compound of Formula (III-f):

or salt thereof.

In certain embodiments, wherein p is 7, the compound of Formula (III) isa compound of Formula (III-g):

or salt thereof.

In certain embodiments, wherein p is 8, the compound of Formula (III) isa compound of Formula (III-h):

or salt thereof.

In certain embodiments, wherein p is 9, the compound of Formula (III) isa compound of Formula (III-i):

or salt thereof.

As generally defined above, each instance of R′ is independentlyhydrogen or optionally substituted alkyl. In certain embodiments, atleast one instance of R′ is hydrogen. In certain embodiments, at leasttwo instances of R′ is hydrogen. In certain embodiments, each instanceof R′ is hydrogen. In certain embodiments, at least one instance of R′is optionally substituted alkyl, e.g., methyl. In certain embodiments,at least two instances of R′ is optionally substituted alkyl, e.g.,methyl. In certain embodiments, one instance of R′ is optionallysubstituted alkyl, and the rest are hydrogen.

As generally defined above, each instance of Q is independently O, S, orNR^(Q), wherein R^(Q) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii), or(iii). In certain embodiments, at least one instance of Q is O. Incertain embodiments, each instance of Q is O. In certain embodiments, atleast one instance of Q is S. In certain embodiments, each instance of Qis S. In certain embodiments, at least one instance of Q is NR^(Z). Incertain embodiments, each instance of Q is NR^(Z). In certainembodiments, each instance of R^(Q) is independently hydrogen or a groupof the formula (i), (ii), or (iii).

As generally defined above, each instance of R¹ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, halogen, —OR^(A1), —N(R^(A1))₂, or—SR^(A1).

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionallysubstituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionallysubstituted C₃₋₄alkyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl,optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl,optionally substituted C₄₋₅alkenyl, or optionally substitutedC₃₋₄alkenyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl,optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl,optionally substituted C₄₋₅alkynyl, or optionally substitutedC₃₋₄alkynyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl,optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted heterocyclyl, e.g., optionally substituted 3-14 memberedheterocyclyl, optionally substituted 3-10 membered heterocyclyl,optionally substituted 5-8 membered heterocyclyl, optionally substituted5-6 membered heterocyclyl, optionally substituted 5 memberedheterocyclyl, or optionally substituted 6 membered heterocyclyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted aryl, e.g., optionally substituted phenyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted heteroaryl, e.g., optionally substituted 5-14 memberedheteroaryl, optionally substituted 5-10 membered heteroaryl, optionallysubstituted 5-6 membered heteroaryl, optionally substituted 5 memberedheteroaryl, or optionally substituted 6 membered heteroaryl.

In any of the above embodiments, the R¹ alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted,for example, with an optionally substituted amino group (e.g., —NR⁶R⁷),an optionally substituted hydroxyl group (e.g., —OR⁶), an optionallysubstituted thiol group (e.g., —SR⁶), or with a group of formula (i),(ii), or (iii), wherein each instance of R⁶ and R⁷ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group whenattached to a nitrogen atom, an oxygen protecting group when attached toan oxygen atom, and a sulfur protecting group when attached to a sulfuratom, or a group of formula (i), (ii), or (iii).

For example, in certain embodiments, at least one instance of R¹ is analkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroarylgroup substituted with an amino group of the formula —N(R⁶)(R⁷). In thatinstance, in certain embodiments, at least one instance of R¹ is a groupof formula:

wherein:

L is an optionally substituted alkylene, optionally substitutedalkenylene, optionally substituted alkynylene, optionally substitutedheteroalkylene, optionally substituted heteroalkenylene, optionallysubstituted heteroalkynylene, optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,or optionally substituted heteroarylene, or combination thereof, and

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, and a nitrogen protecting group;

provided at least one instance of R⁶ and R⁷ is a group of the formula(i), (ii), or (iii):

wherein R′, X, Y, R^(L), and R^(P) are as defined herein.

In certain embodiments, at least two instances of R¹ is a group offormula (iv). In certain embodiments, at least three instances of R¹ isa group of formula (iv). In certain embodiments, at least four instancesof R¹ is a group of formula (iv). In certain embodiments, at least fiveinstances of R¹ is a group of formula (iv). In certain embodiments, atleast six instances of R¹ is a group of formula (iv). In certainembodiments, at least seven instances of R¹ is a group of formula (iv).In certain embodiments, at least eight instances of R¹ is a group offormula (iv). In certain embodiments, at least nine instances of R¹ is agroup of formula (iv). In certain embodiments, each instance of R¹ is agroup of formula (iv).

In certain embodiments, L is an optionally substituted alkylene; e.g.,optionally substituted C₁₋₅₀alkylene, optionally substitutedC₁₋₄₀alkylene, optionally substituted C₁₋₃₀alkylene, optionallysubstituted C₁₋₂₀alkylene, optionally substituted C₄₋₂₀alkylene,optionally substituted C₆₋₂₀alkylene, optionally substitutedC₈₋₂₀alkylene, optionally substituted C₁₀₋₂₀alkylene, optionallysubstituted C₁₋₆alkylene, optionally substituted C₂₋₆alkylene,optionally substituted C₃₋₆alkylene, optionally substitutedC₄₋₆alkylene, optionally substituted C₄₋₅alkylene, or optionallysubstituted C₃₋₄alkylene.

In certain embodiments, L is an optionally substituted alkenylene, e.g.,optionally substituted C₂₋₅₀alkenylene, optionally substitutedC₂₋₄₀alkenylene, optionally substituted C₂₋₃₀alkenylene, optionallysubstituted C₂₋₂₀alkenylene, optionally substituted C₄₋₂₀alkenylene,optionally substituted C₆₋₂₀alkenylene, optionally substitutedC₈₋₂₀alkenylene, optionally substituted C₁₀₋₂₀alkenylene, optionallysubstituted C₂₋₆alkenylene, optionally substituted C₃₋₆alkenylene,optionally substituted C₄₋₆alkenylene, optionally substitutedC₄₋₅alkenylene, or optionally substituted C₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted alkynylene, e.g.,optionally substituted C₂₋₅₀alkynylene, optionally substitutedC₂₋₄₀alkynylene, optionally substituted C₂₋₃₀alkynylene, optionallysubstituted C₂₋₂₀alkynylene, optionally substituted C₄₋₂₀alkynylene,optionally substituted C₆₋₂₀alkynylene, optionally substitutedC₈₋₂₀alkynylene, optionally substituted C₁₋₂₀alkynylene, optionallysubstituted C₂₋₆alkynylene, optionally substituted C₃₋₆alkynylene,optionally substituted C₄₋₆alkynylene, optionally substitutedC₄₋₅alkynylene, or optionally substituted C₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted heteroalkylene;e.g., optionally substituted heteroC₁₋₅₀alkylene, optionally substitutedheteroC₁₋₄₀alkylene, optionally substituted heteroC₁₋₃₀alkylene,optionally substituted heteroC₁₋₂₀alkylene, optionally substitutedheteroC₄₋₂₀alkylene, optionally substituted heteroC₆₋₂₀alkylene,optionally substituted heteroC₈₋₂₀alkylene, optionally substitutedheteroC₁₀₋₂₀alkylene, optionally substituted heteroC₁₋₆alkylene,optionally substituted heteroC₂₋₆alkylene, optionally substitutedheteroC₃₋₆alkylene, optionally substituted heteroC₄₋₆alkylene,optionally substituted heteroC₄₋₅alkylene, or optionally substitutedheteroC₃₋₄alkylene.

In certain embodiments, L is an optionally substituted heteroalkenylene,e.g., optionally substituted heteroC₂₋₅₀alkenylene, optionallysubstituted heteroC₂₋₄₀alkenylene, optionally substitutedheteroC₂₋₃₀alkenylene, optionally substituted heteroC₂₋₂₀alkenylene,optionally substituted heteroC₄₋₂₀alkenylene, optionally substitutedheteroC₆₋₂₀alkenylene, optionally substituted heteroC₈₋₂₀alkenylene,optionally substituted heteroC₁₀₋₂₀alkenylene, optionally substitutedheteroC₂₋₆alkenylene, optionally substituted heteroC₃₋₆alkenylene,optionally substituted heteroC₄₋₆alkenylene, optionally substitutedheteroC₄₋₅alkenylene, or optionally substituted heteroC₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted heteroalkynylene,e.g., optionally substituted heteroC₂₋₅₀alkynylene, optionallysubstituted heteroC₂₋₄₀alkynylene, optionally substitutedheteroC₂₋₃₀alkynylene, optionally substituted heteroC₂₋₂₀alkynylene,optionally substituted heteroC₄₋₂₀alkynylene, optionally substitutedheteroC₆₋₂₀alkynylene, optionally substituted heteroC₈₋₂₀alkynylene,optionally substituted heteroC₁₀₋₂₀alkynylene, optionally substitutedheteroC₂₋₆alkynylene, optionally substituted heteroC₃₋₆alkynylene,optionally substituted heteroC₄₋₆alkynylene, optionally substitutedheteroC₄₋₅alkynylene, or optionally substituted heteroC₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted carbocyclylene,e.g., optionally substituted C₃₋₁₀ carbocyclylene, optionallysubstituted C₅₋₈ carbocyclylene, optionally substituted C₅₋₆carbocyclylene, optionally substituted C₅ carbocyclylene, or optionallysubstituted C₆ carbocyclylene.

In certain embodiments, L is an optionally substituted heterocyclylene,e.g., optionally substituted 3-14 membered heterocyclylene, optionallysubstituted 3-10 membered heterocyclylene, optionally substituted 5-8membered heterocyclylene, optionally substituted 5-6 memberedheterocyclylene, optionally substituted 5 membered heterocyclylene, oroptionally substituted 6 membered heterocyclylene.

In certain embodiments, L is an optionally substituted arylene, e.g.,optionally substituted phenylene.

In certain embodiments, L is an optionally substituted heteroarylene,e.g., optionally substituted 5-14 membered heteroarylene, optionallysubstituted 5-10 membered heteroarylene, optionally substituted 5-6membered heteroarylene, optionally substituted 5 membered heteroarylene,or optionally substituted 6 membered heteroarylene.

For example, in certain embodiments, wherein L is an optionallysubstituted alkylene group, the group of formula (iv) is a group of theformula:

wherein q is an integer between 1 and 50, inclusive.

In certain embodiments, q is an integer between 1 and 40, inclusive. Incertain embodiments, q is an integer between 1 and 30, inclusive. Incertain embodiments, q is an integer between 1 and 20, inclusive. Incertain embodiments, q is an integer between 4 and 20, inclusive. Incertain embodiments, q is an integer between 6 and 20, inclusive. Incertain embodiments, q is an integer between 8 and 20, inclusive. Incertain embodiments, q is 1. In certain embodiments, q is 2. In certainembodiments, q is 3. In certain embodiments, q is 4. In certainembodiments, q is 5. In certain embodiments, q is 6. In certainembodiments, q is 7. In certain embodiments, q is 8. In certainembodiments, q is 9. In certain embodiments, q is 10.

In certain embodiments, both R⁶ and R⁷ are hydrogen. In certainembodiments, R⁶ is hydrogen and R⁷ is a group of the formula (i), (ii),or (iii). In certain embodiments, R⁶ is hydrogen and R⁷ is a group ofthe formula (i). In certain embodiments, R⁶ is hydrogen and R⁷ is agroup of the formula (ii). In certain embodiments, R⁶ is hydrogen and R⁷is a group of the formula (iii). In certain embodiments, both R⁶ and R⁷are independently a group of the formula (i), (ii), or (iii). In certainembodiments, both R⁶ and R⁷ are independently a group of the formula(i). In certain embodiments, both R⁶ and R⁷ are independently a group ofthe formula (ii). In certain embodiments, both R⁶ and R⁷ areindependently a group of the formula (iii). In certain embodiments, bothR⁶ and R⁷ are the same group, selected from a group of the formula (i),(ii), or (iii).

It is understood that R¹ encompasses amino acid side chains such asexemplified in Table 1 of the Examples. In certain embodiments, R¹ is agroup selected from any one of the amino acid side chain groups listedtherein.

In certain embodiments, each instance of R¹ is the same. In certainembodiments, at least one R¹ group is different. In certain embodiments,each R¹ group is different.

As generally defined above, each instance of R² is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group, or agroup of the formula (i), (ii), or (iii):

wherein R′, X, Y, R^(L), and R^(P) are as defined herein.

In certain embodiments, at least one instance of R² is optionallysubstituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionallysubstituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionallysubstituted C₃₋₄alkyl.

In certain embodiments, at least one instance of R² is optionallysubstituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl,optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl,optionally substituted C₄₋₅alkenyl, or optionally substitutedC₃₋₄alkenyl.

In certain embodiments, at least one instance of R² is optionallysubstituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl,optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl,optionally substituted C₄₋₅alkynyl, or optionally substitutedC₃₋₄alkynyl.

In certain embodiments, at least one instance of R² is optionallysubstituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl,optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl.

In certain embodiments, at least one instance of R² is optionallysubstituted heterocyclyl, e.g., optionally substituted 3-14 memberedheterocyclyl, optionally substituted 3-10 membered heterocyclyl,optionally substituted 5-8 membered heterocyclyl, optionally substituted5-6 membered heterocyclyl, optionally substituted 5 memberedheterocyclyl, or optionally substituted 6 membered heterocyclyl.

In certain embodiments, at least one instance of R² is optionallysubstituted aryl, e.g., optionally substituted phenyl.

In certain embodiments, at least one instance of R² is optionallysubstituted heteroaryl, e.g., optionally substituted 5-14 memberedheteroaryl, optionally substituted 5-10 membered heteroaryl, optionallysubstituted 5-6 membered heteroaryl, optionally substituted 5 memberedheteroaryl, or optionally substituted 6 membered heteroaryl.

In certain embodiments, at least one instance of R² is a nitrogenprotecting group.

In certain embodiments, at least one instance of R² is a group of theformula (i). In certain embodiments, at least one instance of R² is agroup of the formula (ii). In certain embodiments, at least one instanceof R² is a group of the formula (iii).

In certain embodiments, each instance of R² is a group other thanformula (i), (ii), or (iii); in that instance, it follows that at leastone R^(Q) is a group of the formula (i), (ii), or (iii), or at least oneR¹ is a group of formula (iv), and at least one of R⁶ or R⁷ encompassedby R¹ is a group of the formula (i), (ii), or (iii). For example, incertain embodiments, both instances of R² are hydrogen, and thus atleast one R^(Q) is a group of the formula (i), (ii), or (iii), or atleast one R¹ is a group of formula (iv), and at least one of R⁶ or R⁷encompassed by R¹ is a group of the formula (i), (ii), or (iii).

Various combinations of the above embodiments of Formula (III) arecontemplated herein.

For example, in certain embodiments, wherein each instance of Q is 0,the compound of Formula (III) is a compound of Formula (III-a):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, each instance of R¹ is a group offormula (iv). In certain embodiments, each instance of R² is hydrogen.In certain embodiments, at least one instance of R² is a group offormula (i). In certain embodiments, at least one instance of R² is agroup of formula (ii). In certain embodiments, at least one instance ofR² is a group of formula (iii). In certain embodiments, p is 1. Incertain embodiments, p is 2. In certain embodiments, p is 3.

In certain embodiments of Formula (III-a), wherein at least one R¹ is agroup the formula (iv), provided is a compound of Formula (III-b):

or salt thereof. In certain embodiments, each instance of R¹ is a groupof formula (iv). In certain embodiments, R² is hydrogen. In certainembodiments, each instance of R² is hydrogen. In certain embodiments, atleast one instance of R² is a group of formula (i). In certainembodiments, at least one instance of R² is a group of formula (ii). Incertain embodiments, at least one instance of R² is a group of formula(iii). In certain embodiments, p is 1. In certain embodiments, p is 2.In certain embodiments, p is 3. In certain embodiments, L is anoptionally substituted alkylene. In certain embodiments, R⁶ is a groupof formula (i). In certain embodiments, R⁶ is a group of formula (ii).In certain embodiments, R⁶ is a group of formula (iii). In certainembodiments, R⁷ is a group of formula (i). In certain embodiments, R⁷ isa group of formula (ii). In certain embodiments, R⁷ is a group offormula (iii). In certain embodiments, both R⁶ and R⁷ are independentlygroups of formula (i), (ii), or (iii).

In certain embodiments of Formula (III-a), wherein each instance of R¹is a group the formula (iv), provided is a compound of Formula (III-c):

or salt thereof. In certain embodiments, each instance of R² ishydrogen. In certain embodiments, at least one instance of R² is a groupof formula (i). In certain embodiments, at least one instance of R² is agroup of formula (ii). In certain embodiments, at least one instance ofR² is a group of formula (iii). In certain embodiments, p is 1. Incertain embodiments, p is 2. In certain embodiments, p is 3. In certainembodiments, L is an optionally substituted alkylene. In certainembodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ isa group of formula (ii). In certain embodiments, R⁶ is a group offormula (iii). In certain embodiments, R⁷ is a group of formula (i). Incertain embodiments, R⁷ is a group of formula (ii). In certainembodiments, R⁷ is a group of formula (iii). In certain embodiments,both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii).

In certain embodiments of Formula (III-c), wherein p is 1, provided is acompound of Formula (III-c1):

or salt thereof. In certain embodiments, each instance of R² ishydrogen. In certain embodiments, at least one instance of R² is a groupof formula (i). In certain embodiments, at least one instance of R² is agroup of formula (ii). In certain embodiments, at least one instance ofR² is a group of formula (iii). In certain embodiments, L is anoptionally substituted alkylene. In certain embodiments, R⁶ is a groupof formula (i). In certain embodiments, R⁶ is a group of formula (ii).In certain embodiments, R⁶ is a group of formula (iii). In certainembodiments, R⁷ is a group of formula (i). In certain embodiments, R⁷ isa group of formula (ii). In certain embodiments, R⁷ is a group offormula (iii). In certain embodiments, both R⁶ and R⁷ are independentlygroups of formula (i), (ii), or (iii).

In certain embodiments of Formula (III-c1), wherein each instance of R²is hydrogen, provided is a compound of Formula (III-c2):

or salt thereof. In certain embodiments, L is an optionally substitutedalkylene. In certain embodiments, R⁶ is a group of formula (i). Incertain embodiments, R⁶ is a group of formula (ii). In certainembodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷is a group of formula (i). In certain embodiments, R⁷ is a group offormula (ii). In certain embodiments, R⁷ is a group of formula (iii). Incertain embodiments, both R⁶ and R⁷ are groups of formula (i), (ii), or(iii).

In certain embodiments of Formula (III-c1), wherein L is an optionallysubstituted alkylene, provided is a compound of Formula (III-c3):

or salt thereof, wherein q is an integer between 1 and 10, inclusive. Incertain embodiments, R⁶ is a group of formula (i). In certainembodiments, R⁶ is a group of formula (ii). In certain embodiments, R⁶is a group of formula (iii). In certain embodiments, R⁷ is a group offormula (i). In certain embodiments, R⁷ is a group of formula (ii). Incertain embodiments, R⁷ is a group of formula (iii). In certainembodiments, both R⁶ and R⁷ are independently groups of formula (i),(ii), or (iii).

In certain embodiments of Formula (III-a), wherein at least one instanceof R² is a group of formula (i) and each instance of R′ is hydrogen,provided is a compound of Formula (III-d):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, each instance of R¹ is a group offormula (iv). In certain embodiments, each instance of R² is hydrogen.In certain embodiments, at least one instance of R² is a group offormula (i). In certain embodiments, at least one instance of R² is agroup of formula (ii). In certain embodiments, at least one instance ofR² is a group of formula (iii). In certain embodiments, R² is a group offormula (iii). In certain embodiments, p is 1. In certain embodiments, pis 2. In certain embodiments, p is 3.

In certain embodiments of Formula (III-a), wherein at least one instanceof R² is a group of formula (ii) and each instance of R′ is hydrogen,provided is a compound of Formula (III-e):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, each instance of R¹ is a group offormula (iv). In certain embodiments, each instance of R² is hydrogen.In certain embodiments, at least one instance of R² is a group offormula (i). In certain embodiments, at least one instance of R² is agroup of formula (ii). In certain embodiments, at least one instance ofR² is a group of formula (iii). In certain embodiments, p is 1. Incertain embodiments, p is 2. In certain embodiments, p is 3.

In certain embodiments of Formula (III-a), wherein at least one instanceof R² is a group of formula (iii), provided is a compound of Formula(III-f):

or salt thereof. In certain embodiments, at least one R¹ is a group offormula (iv). In certain embodiments, each instance of R¹ is a group offormula (iv). In certain embodiments, each instance of R² is hydrogen.In certain embodiments, at least one instance of R² is a group offormula (i). In certain embodiments, at least one instance of R² is agroup of formula (ii). In certain embodiments, at least one instance ofR² is a group of formula (iii). In certain embodiments, p is 1. Incertain embodiments, p is 2. In certain embodiments, p is 3.

Compounds of Formula (IV), (V), and (VI)

Compounds of Formula (IV), (V), and (VI), while not constructed fromamino acid starting materials, share the same molecular formula andcyclic motif, and are thus structural isomers of compounds of Formula(III-a). The present invention embraces each as exemplary APPLstructural isomers of the present invention.

Thus, in yet another aspect, provided is a compound of Formula (IV),(V), or (VI):

or salt thereof;wherein:

each instance of Q is independently O, S, or NR^(Q), wherein R^(Q) ishydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group, or agroup of formula (i), (ii), (iii);

each instance of R¹ is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, halogen,—OR^(A1), —N(R^(A1))₂, or —SR^(A1); wherein each occurrence of R^(A1) isindependently hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, an oxygenprotecting group when attached to an oxygen atom, a sulfur protectinggroup when attached to an sulfur atom, a nitrogen protecting group whenattached to a nitrogen atom, or two R^(A1) groups are joined to form anoptionally substituted heterocyclic or optionally substituted heteroarylring;

each instance of R² is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of formula (i), (ii), or (iii);and

Formulae (i), (ii), and (iii) are:

wherein:

each instance of R′ is independently hydrogen or optionally substitutedalkyl;

X is O, S, NR^(X), wherein R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

Y is O, S, NR^(Y), wherein R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

R^(P) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached to asulfur atom, or a nitrogen protecting group when attached to a nitrogenatom; and

R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substitutedC₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionallysubstituted heteroC₁₋₅₀alkyl, optionally substituted heteroC₂₋₅₀alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer;

provided that at least one instance of R^(Q), R², R⁶, or R⁷ is a groupof the formula (i), (ii), or (iii).

As generally defined above, each instance of Q is independently O, S, orNR^(Q), wherein R^(Q) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii), or(iii). In certain embodiments, at least one instance of Q is O. Incertain embodiments, each instance of Q is O. In certain embodiments, atleast one instance of Q is S. In certain embodiments, each instance of Qis S. In certain embodiments, at least one instance of Q is NR^(Z). Incertain embodiments, each instance of Q is NR^(Z). In certainembodiments, each instance of R^(Q) is independently hydrogen or a groupof the formula (i), (ii), or (iii).

As generally defined above, each instance of R′ is independentlyhydrogen or optionally substituted alkyl. In certain embodiments, atleast one instance of R′ is hydrogen. In certain embodiments, at leasttwo instances of R′ is hydrogen. In certain embodiments, each instanceof R′ is hydrogen. In certain embodiments, at least one instance of R′is optionally substituted alkyl, e.g., methyl. In certain embodiments,at least two instances of R′ is optionally substituted alkyl, e.g.,methyl. In certain embodiments, one instance of R′ is optionallysubstituted alkyl, and the rest are hydrogen.

As generally defined above, each instance of R¹ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, halogen, —OR^(A1), —N(R^(A1))₂, or—SR^(A1).

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionallysubstituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionallysubstituted C₃₋₄alkyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl,optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl,optionally substituted C₄₋₅alkenyl, or optionally substitutedC₃₋₄alkenyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl,optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl,optionally substituted C₄₋₅alkynyl, or optionally substitutedC₃₋₄alkynyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl,optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted heterocyclyl, e.g., optionally substituted 3-14 memberedheterocyclyl, optionally substituted 3-10 membered heterocyclyl,optionally substituted 5-8 membered heterocyclyl, optionally substituted5-6 membered heterocyclyl, optionally substituted 5 memberedheterocyclyl, or optionally substituted 6 membered heterocyclyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted aryl, e.g., optionally substituted phenyl.

In certain embodiments, at least one instance of R¹ is optionallysubstituted heteroaryl, e.g., optionally substituted 5-14 memberedheteroaryl, optionally substituted 5-10 membered heteroaryl, optionallysubstituted 5-6 membered heteroaryl, optionally substituted 5 memberedheteroaryl, or optionally substituted 6 membered heteroaryl.

In any of the above embodiments, the R¹ alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted,for example, with an optionally substituted amino group (e.g., —NR⁶R⁷),an optionally substituted hydroxyl group (e.g., —OR⁶), an optionallysubstituted thiol group (e.g., —SR⁶), or with a group of formula (i),(ii), or (iii), wherein each instance of R⁶ and R⁷ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group whenattached to a nitrogen atom, an oxygen protecting group when attached toan oxygen atom, and a sulfur protecting group when attached to a sulfuratom, or a group of formula (i), (ii), or (iii).

For example, in certain embodiments, at least one instance of R¹ is analkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroarylgroup substituted with an amino group of the formula —N(R⁶)(R⁷). In thatinstance, in certain embodiments, at least one instance of R¹ is a groupof formula:

wherein:

L is an optionally substituted alkylene, optionally substitutedalkenylene, optionally substituted alkynylene, optionally substitutedheteroalkylene, optionally substituted heteroalkenylene, optionallysubstituted heteroalkynylene, optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,or optionally substituted heteroarylene, or combination thereof, and

R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, and a nitrogen protecting group;

provided at least one instance of R⁶ and R⁷ is a group of the formula(i), (ii), or (iii):

wherein R′, X, Y, R^(L), and R^(P) are as defined herein.

In certain embodiments, both instances of R¹ are groups of formula (iv).

In certain embodiments, L is an optionally substituted alkylene; e.g.,optionally substituted C₁₋₅₀alkylene, optionally substitutedC₁₋₄₀alkylene, optionally substituted C₁₋₃₀alkylene, optionallysubstituted C₁₋₂₀alkylene, optionally substituted C₄₋₂₀alkylene,optionally substituted C₆₋₂₀alkylene, optionally substitutedC₈₋₂₀alkylene, optionally substituted C₁₀₋₂₀alkylene, optionallysubstituted C₁₋₆alkylene, optionally substituted C₂₋₆alkylene,optionally substituted C₃₋₆alkylene, optionally substitutedC₄₋₆alkylene, optionally substituted C₄₋₅alkylene, or optionallysubstituted C₃₋₄alkylene.

In certain embodiments, L is an optionally substituted alkenylene, e.g.,optionally substituted C₂₋₅₀alkenylene, optionally substitutedC₂₋₄₀alkenylene, optionally substituted C₂₋₃₀alkenylene, optionallysubstituted C₂₋₂₀alkenylene, optionally substituted C₄₋₂₀alkenylene,optionally substituted C₆₋₂₀alkenylene, optionally substitutedC₈₋₂₀alkenylene, optionally substituted C₁₀₋₂₀alkenylene, optionallysubstituted C₂₋₆alkenylene, optionally substituted C₃₋₆alkenylene,optionally substituted C₄₋₆alkenylene, optionally substitutedC₄₋₅alkenylene, or optionally substituted C₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted alkynylene, e.g.,optionally substituted C₂₋₅₀alkynylene, optionally substitutedC₂₋₄₀alkynylene, optionally substituted C₂₋₃₀alkynylene, optionallysubstituted C₂₋₂₀alkynylene, optionally substituted C₄₋₂₀alkynylene,optionally substituted C₆₋₂₀alkynylene, optionally substitutedC₈₋₂₀alkynylene, optionally substituted C₁₀₋₂₀alkynylene, optionallysubstituted C₂₋₆alkynylene, optionally substituted C₃₋₆alkynylene,optionally substituted C₄₋₆alkynylene, optionally substitutedC₄₋₅alkynylene, or optionally substituted C₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted heteroalkylene;e.g., optionally substituted heteroC₁₋₅₀alkylene, optionally substitutedheteroC₁₋₄₀alkylene, optionally substituted heteroC₁₋₃₀alkylene,optionally substituted heteroC₁₋₂₀alkylene, optionally substitutedheteroC₄₋₂₀alkylene, optionally substituted heteroC₆₋₂₀alkylene,optionally substituted heteroC₈₋₂₀alkylene, optionally substitutedheteroC₁₀₋₂₀alkylene, optionally substituted heteroC₁₋₆alkylene,optionally substituted heteroC₂₋₆alkylene, optionally substitutedheteroC₃₋₆alkylene, optionally substituted heteroC₄₋₆alkylene,optionally substituted heteroC₄₋₅alkylene, or optionally substitutedheteroC₃₋₄alkylene.

In certain embodiments, L is an optionally substituted heteroalkenylene,e.g., optionally substituted heteroC₂₋₅₀alkenylene, optionallysubstituted heteroC₂₋₄₀alkenylene, optionally substitutedheteroC₂₋₃₀alkenylene, optionally substituted heteroC₂₋₂₀alkenylene,optionally substituted heteroC₄₋₂₀alkenylene, optionally substitutedheteroC₆₋₂₀alkenylene, optionally substituted heteroC₈₋₂₀alkenylene,optionally substituted heteroC₁₀₋₂₀alkenylene, optionally substitutedheteroC₂₋₆alkenylene, optionally substituted heteroC₃₋₆alkenylene,optionally substituted heteroC₄₋₆alkenylene, optionally substitutedheteroC₄₋₅alkenylene, or optionally substituted heteroC₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted heteroalkynylene,e.g., optionally substituted heteroC₂₋₅₀alkynylene, optionallysubstituted heteroC₂₋₄₀alkynylene, optionally substitutedheteroC₂₋₃₀alkynylene, optionally substituted heteroC₂₋₂₀alkynylene,optionally substituted heteroC₄₋₂₀alkynylene, optionally substitutedheteroC₆₋₂₀alkynylene, optionally substituted heteroC₈₋₂₀alkynylene,optionally substituted heteroC₁₀₋₂₀alkynylene, optionally substitutedheteroC₂₋₆alkynylene, optionally substituted heteroC₃₋₆alkynylene,optionally substituted heteroC₄₋₆alkynylene, optionally substitutedheteroC₄₋₅alkynylene, or optionally substituted heteroC₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted carbocyclylene,e.g., optionally substituted C₃₋₁₀ carbocyclylene, optionallysubstituted C₅₋₈ carbocyclylene, optionally substituted C₅₋₆carbocyclylene, optionally substituted C₅ carbocyclylene, or optionallysubstituted C₆ carbocyclylene.

In certain embodiments, L is an optionally substituted heterocyclylene,e.g., optionally substituted 3-14 membered heterocyclylene, optionallysubstituted 3-10 membered heterocyclylene, optionally substituted 5-8membered heterocyclylene, optionally substituted 5-6 memberedheterocyclylene, optionally substituted 5 membered heterocyclylene, oroptionally substituted 6 membered heterocyclylene.

In certain embodiments, L is an optionally substituted arylene, e.g.,optionally substituted phenylene.

In certain embodiments, L is an optionally substituted heteroarylene,e.g., optionally substituted 5-14 membered heteroarylene, optionallysubstituted 5-10 membered heteroarylene, optionally substituted 5-6membered heteroarylene, optionally substituted 5 membered heteroarylene,or optionally substituted 6 membered heteroarylene.

For example, in certain embodiments, wherein L is an optionallysubstituted alkylene group, the group of formula (iv) is a group of theformula:

wherein q is an integer between 1 and 50, inclusive.

In certain embodiments, q is an integer between 1 and 40, inclusive. Incertain embodiments, q is an integer between 1 and 30, inclusive. Incertain embodiments, q is an integer between 1 and 20, inclusive. Incertain embodiments, q is an integer between 4 and 20, inclusive. Incertain embodiments, q is an integer between 6 and 20, inclusive. Incertain embodiments, q is an integer between 8 and 20, inclusive. Incertain embodiments, q is 1. In certain embodiments, q is 2. In certainembodiments, q is 3. In certain embodiments, q is 4. In certainembodiments, q is 5. In certain embodiments, q is 6. In certainembodiments, q is 7. In certain embodiments, q is 8. In certainembodiments, q is 9. In certain embodiments, q is 10.

In certain embodiments, both R⁶ and R⁷ are hydrogen. In certainembodiments, R⁶ is hydrogen and R⁷ is a group of the formula (i), (ii),or (iii). In certain embodiments, R⁶ is hydrogen and R⁷ is a group ofthe formula (i). In certain embodiments, R⁶ is hydrogen and R⁷ is agroup of the formula (ii). In certain embodiments, R⁶ is hydrogen and R⁷is a group of the formula (iii). In certain embodiments, both R⁶ and R⁷are independently a group of the formula (i), (ii), or (iii). In certainembodiments, both R⁶ and R⁷ are independently a group of the formula(i). In certain embodiments, both R⁶ and R⁷ are independently a group ofthe formula (ii). In certain embodiments, both R⁶ and R⁷ areindependently a group of the formula (iii). In certain embodiments, bothR⁶ and R⁷ are the same group, selected from a group of the formula (i),(ii), or (iii).

It is understood that R¹ encompasses amino acid side chains such asexemplified in Table 1 of the Examples. In certain embodiments, R¹ is agroup selected from any one of the amino acid side chain groups listedtherein.

In certain embodiments, each instance of R¹ is the same. In certainembodiments, at least one R¹ group is different. In certain embodiments,each R¹ group is different.

As generally defined above, each instance of R² is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group, or agroup of the formula (i), (ii), or (iii):

wherein R′, X, Y, R^(L), and R^(P) are as defined herein.

In certain embodiments, at least one instance of R² is optionallysubstituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionallysubstituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionallysubstituted C₃₋₄alkyl.

In certain embodiments, at least one instance of R² is optionallysubstituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl,optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl,optionally substituted C₄₋₅alkenyl, or optionally substitutedC₃₋₄alkenyl.

In certain embodiments, at least one instance of R² is optionallysubstituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl,optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl,optionally substituted C₄₋₅alkynyl, or optionally substitutedC₃₋₄alkynyl.

In certain embodiments, at least one instance of R² is optionallysubstituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl,optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl.

In certain embodiments, at least one instance of R² is optionallysubstituted heterocyclyl, e.g., optionally substituted 3-14 memberedheterocyclyl, optionally substituted 3-10 membered heterocyclyl,optionally substituted 5-8 membered heterocyclyl, optionally substituted5-6 membered heterocyclyl, optionally substituted 5 memberedheterocyclyl, or optionally substituted 6 membered heterocyclyl.

In certain embodiments, at least one instance of R² is optionallysubstituted aryl, e.g., optionally substituted phenyl.

In certain embodiments, at least one instance of R² is optionallysubstituted heteroaryl, e.g., optionally substituted 5-14 memberedheteroaryl, optionally substituted 5-10 membered heteroaryl, optionallysubstituted 5-6 membered heteroaryl, optionally substituted 5 memberedheteroaryl, or optionally substituted 6 membered heteroaryl.

In certain embodiments, at least one instance of R² is a nitrogenprotecting group.

In certain embodiments, at least one instance of R² is a group of theformula (i). In certain embodiments, at least one instance of R² is agroup of the formula (ii). In certain embodiments, at least one instanceof R² is a group of the formula (iii).

In certain embodiments, each instance of R² is a group other thanformula (i), (ii), or (iii); in that instance, it follows that at leastone R^(Q) is a group of the formula (i), (ii), or (iii), or at least oneR¹ is a group of formula (iv), and at least one of R⁶ or R⁷ encompassedby R¹ is a group of the formula (i), (ii), or (iii). For example, incertain embodiments, both instances of R² are hydrogen, and thus atleast one R^(Q) is a group of the formula (i), (ii), or (iii), or atleast one R¹ is a group of formula (iv), and at least one of R⁶ or R⁷encompassed by R¹ is a group of the formula (i), (ii), or (iii).

Various combinations of the above embodiments of Formula (IV), (V), and(VI) are contemplated herein. For example, in certain embodiments,wherein each instance of Q is O, the compound of Formula (IV), (V), or(VI) is a compound of Formula (IV-a), (V-a), or (VI-a):

or salt thereof. In certain embodiments, at least one instance of R¹ isa group of formula (iv). In certain embodiments, each instance of R¹ isa group of formula (iv). In certain embodiments, at least one instanceof R² is optionally substituted alkyl, optionally substituted alkenyl,or optionally substituted alkynyl. In certain embodiments, at least oneinstance of R² is a group of formula (i). In certain embodiments, atleast one instance of R² is a group of formula (ii). In certainembodiments, at least one instance of R² is a group of formula (iii).

In certain embodiments of Formula (IV-a), (V-a), or (VI-a), wherein atleast one R¹ is a group the formula (iv), provided is a compound ofFormula (IV-b), (V-b), or (VI-b):

or salt thereof. In certain embodiments, at least one instance of R² isoptionally substituted alkyl, optionally substituted alkenyl, oroptionally substituted alkynyl. In certain embodiments, at least oneinstance of R² is a group of formula (i). In certain embodiments, atleast one instance of R² is a group of formula (ii). In certainembodiments, at least one instance of R² is a group of formula (iii). Incertain embodiments, L is an optionally substituted alkylene. In certainembodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ isa group of formula (ii). In certain embodiments, R⁶ is a group offormula (iii). In certain embodiments, R⁷ is a group of formula (i). Incertain embodiments, R⁷ is a group of formula (ii). In certainembodiments, R⁷ is a group of formula (iii). In certain embodiments,both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii).

In certain embodiments of Formula (IV-b), (V-b), or (VI-b), wherein bothR¹ groups are a group the formula (iv), provided is a compound ofFormula (IV-c), (V-c), or (VI-c):

or salt thereof. In certain embodiments, at least one instance of R² isoptionally substituted alkyl, optionally substituted alkenyl, oroptionally substituted alkynyl. In certain embodiments, at least oneinstance of R² is a group of formula (i). In certain embodiments, atleast one instance of R² is a group of formula (ii). In certainembodiments, at least one instance of R² is a group of formula (iii). Incertain embodiments, L is an optionally substituted alkylene. In certainembodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ isa group of formula (ii). In certain embodiments, R⁶ is a group offormula (iii). In certain embodiments, R⁷ is a group of formula (i). Incertain embodiments, R⁷ is a group of formula (ii). In certainembodiments, R⁷ is a group of formula (iii). In certain embodiments,both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii).

In certain embodiments of Formulae (IV-a), (V-a), and (VI-a), wherein atleast one instance of R² is a group of formula (i) and each instance ofR′ is hydrogen, provided is a compound of Formulae (IV-d), (V-d), and(VI-d):

or salt thereof. In certain embodiments, at least one instance of R¹ isa group of formula (iv). In certain embodiments, each instance of R¹ isa group of formula (iv). In certain embodiments, R² is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted alkynyl. In certain embodiments, R² is a group of formula(i). In certain embodiments, R² is a group of formula (ii). In certainembodiments, R² is a group of formula (iii).

In certain embodiments of Formulae (IV-a), (V-a), and (VI-a), whereinboth instances of R² is a group of formula (i) and each instance of R′is hydrogen, provided is a compound of Formulae (IV-e), (V-e), and(VI-e):

or salt thereof. In certain embodiments, at least one instance of R¹ isa group of formula (iv). In certain embodiments, each instance of R¹ isa group of formula (iv).

In certain embodiments of Formulae (IV-a), (V-a), and (VI-a), wherein atleast one instance of R² is a group of formula (ii) and each instance ofR′ is hydrogen, provided is a compound of Formulae (IV-f), (V-f), and(VI-f):

or salt thereof. In certain embodiments, at least one instance of R is agroup of formula (iv). In certain embodiments, each instance of R¹ is agroup of formula (iv). In certain embodiments, R² is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted alkynyl. In certain embodiments, R² is a group of formula(i). In certain embodiments, R² is a group of formula (ii). In certainembodiments, R² is a group of formula (iii).

In certain embodiments of Formulae (IV-a), (V-a), and (VI-a), whereinboth instances of R² is a group of formula (ii) and each instance of R′is hydrogen, provided is a compound of Formulae (IV-g), (V-g), and(VI-g):

or salt thereof. In certain embodiments, at least one instance of R is agroup of formula (iv). In certain embodiments, each instance of R¹ is agroup of formula (iv).

In certain embodiments of Formulae (IV-a), (V-a), and (VI-a), wherein atleast one instance of R² is a group of formula (iii), provided is acompound of Formulae (IV-h), (V-h), and (VI-h):

or salt thereof. In certain embodiments, at least one instance of R¹ isa group of formula (iv). In certain embodiments, each instance of R¹ isa group of formula (iv). In certain embodiments, R² is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted alkynyl. In certain embodiments, R² is a group of formula(i). In certain embodiments, R² is a group of formula (ii). In certainembodiments, R² is a group of formula (iii).

In certain embodiments of Formulae (IV-a), (V-a), and (VI-a), whereinboth instances of R² are a group of formula (iii), provided is acompound of Formulae (IV-e), (V-e), and (VI-e):

or salt thereof. In certain embodiments, at least one instance of R¹ isa group of formula (iv). In certain embodiments, each instance of R¹ isa group of formula (iv).Groups of Formula (i), (ii), and (iii)

As understood from the above discussion, APPLs, and in particular, APPLcompounds of Formulae (I), (III), (IV), (V), and (VI), each include atleast one instance of a group of the formula (i), (ii), or (iii):

wherein:

each instance of R′ is independently hydrogen or optionally substitutedalkyl;

X is O, S, NR^(X), wherein R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

Y is O, S, NR^(Y), wherein R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group;

R^(P) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached to asulfur atom, or a nitrogen protecting group when attached to a nitrogenatom; and

R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substitutedC₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionallysubstituted heteroC₁₋₅₀alkyl, optionally substituted heteroC₂₋₅₀alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.

In the case of Formula (II), the at least one instance of group offormula (i) is incorporated as part of the scaffold, e.g., bymonoaddition of a compound (i-x), followed by internal cyclization. See,e.g., Scheme 2.

In certain embodiments, an APPL, and in particular, a compound ofFormulae (I), (II), (III), (IV), (V), or (VI), comprises at least oneinstance of a group of the formula (i) attached thereto:

In certain embodiments of formula (i), Y is O. In certain embodiments offormula (i), Y is S. In certain embodiments of formula (i), Y is NR^(Y),wherein R^(Y) is hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, or a nitrogenprotecting group. In certain embodiments of formula (i), Y is NR^(Y),wherein R^(Y) is hydrogen, optionally substituted alkyl, or a nitrogenprotecting group. In certain embodiments of formula (i), each instanceof R′ is hydrogen.

As used herein, when the group R^(L) is depicted as bisecting acarbon-carbon bond, e.g., of the group of the formula (i), it isunderstood that R^(L) may be substituted at either carbon. Nucleophilicattack of an amino or amide group at the least sterically hinderedcarbon of the epoxide, thiirane, or aziridine of formula (i-x) providesa group of the formula (i-a1), (i-a2), or (i-a3) (route a), whilenucleophilic attack at the more sterically hindered carbon of theepoxide, thiirane, or aziridine of formula (i-x) provides a group of theformula (i-b1), (i-b2), or (i-b3) (route b), wherein R^(P) is hydrogen(Scheme 6). It is understood that compounds of the present invention maycomprise a mixture of products attached thereto arising from route (a)and route (b) depending on the preference, or lack thereof, of the modeof addition. The bisecting group R^(L) depicted in the Formulae seeks toencompasses all contemplated modes of addition.

The resulting hydroxyl, thiol, or amino group —YR^(P), wherein R^(P) ishydrogen, may optionally be converted to a substituted group, whereinR^(P) is a group other than hydrogen, i.e., wherein R^(P) isindependently selected from optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, an oxygenprotecting group when attached to an oxygen atom, a sulfur protectinggroup when attached to a sulfur atom, or a nitrogen protecting groupwhen attached to a nitrogen atom; using conventional methods.Alkylation, acylation, and/or protection of a hydroxyl, thiol, or aminomoiety are methods well-known in the art; see, e.g., Protecting Groupsin Organic Synthesis, T. W. Greene and P. G. M. Wuts, ^(3rd) edition,John Wiley & Sons, 1999; Smith and March, March's Advanced OrganicChemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; and Carruthers, Some Modern Methods of Organic Synthesis,3^(rd) Edition, Cambridge University Press, Cambridge, 1987. Forexample, in certain non-limiting embodiments, the hydroxyl, thiol, oramino moiety —YR^(P), wherein R^(P) is hydrogen, may be reacted with anelectrophile of the formula R^(P)—X² wherein R^(P) is a group other thanhydrogen, and X² is a leaving group, to provide a substituted hydroxyl,thiol, and amino group in formula (i).

In certain embodiments of formula (i), R^(P) is hydrogen. In certainembodiments of formula (i), R^(P) is optionally substituted alkyl. Incertain embodiments of formula (i), R^(P) is optionally substitutedalkenyl. In certain embodiments of formula (i), R^(P) is optionallysubstituted alkynyl. In certain embodiments of formula (i), R^(P) isoptionally substituted carbocyclyl. In certain embodiments of formula(i), R^(P) is optionally substituted heterocyclyl. In certainembodiments of formula (i), R^(P) is optionally substituted aryl. Incertain embodiments of formula (i), R^(P) is optionally substitutedheteroaryl. In certain embodiments of formula (i), R^(P) is an oxygenprotecting group when attached to an oxygen atom. In certain embodimentsof formula (i), R^(P) is a sulfur protecting group when attached to asulfur atom. In certain embodiments of formula (i), R^(P) is a nitrogenprotecting group when attached to a nitrogen atom.

It is understood from the present disclosure that the group of formula(i) represents a group of formula (i-a) or a group of formula (i-b):

In certain embodiments, the reaction mixture provides a mixture of APPLscomprising more APPLs conjugated to a group of formula (i-a) thanformula (i-b), e.g., the reaction mixture comprises greater than 50%,greater than 60%, greater than 70%, greater than 80%, greater than 90%,greater than 95%, greater than 99%, between about 60% to about 100%,between about 70% to about 100%, between about 80% to about 100%,between about 90% to about 100%, between about 95% to about 100%, orbetween about 99% to about 100%, of an APPL attached to formula (i-a).

In certain embodiments, the epoxide, thiirane, or aziridine of formula(i-x) is chiral, i.e., having (R) or (S) stereochemistry. Chiralepoxides, thiiranes, and aziridines can be obtained from a variety ofsources which are familiar to those skilled in the art of organicsynthesis. In some embodiments, the chiral epoxide, thiirane, oraziridine is obtained commercially. In some embodiments, the chiralepoxide, thiirane, or aziridine is synthesized according to methodsknown to those of skill in the art, such as, but not limited to theSharpless epoxidation of primary and secondary allylic alcohols into2,3-epoxyalcohols (see, e.g., Katsuki et al., J. Am. Chem. Soc. 1980,102, 5974; Hill et al., Org. Syn., Coll. Vol. 7, p. 461 (1990); Vol. 63,p. 66 (1985); Katsuki et al., Org. React. 1996, 48, 1-300). In someembodiments, the chiral epoxide, thiirane, or aziridine is obtained fromthe resolution of a mixture (e.g., racemic mixture) of epoxides,thiiranes, or aziridines. In some embodiments, the chiral epoxide,thiirane, or aziridine is obtained by the separation of enantiomers ordiastereoisomers using chiral chromatography. Chirality can becharacterized in a variety of ways, e.g., obtaining a crystal structureof the compound containing a heavy atom attached thereto, obtaining theoptical rotation of the compound, and/or NMR analysis after chemicalmodification of the optically active compound with a chiral derivatizingagent are some methods useful in evaluating chirality.

In certain embodiments, wherein the epoxide, thiirane, or aziridine offormula (i-x1) is chiral, the conjugation reaction is regioselective,and the reaction provides a chiral mixture of APPLs comprising moreAPPLs conjugated to a group of formula (i-a1) than formula (i-b1), e.g.,the reaction mixture comprises greater than 50%, greater than 60%,greater than 70%, greater than 80%, greater than 90%, greater than 95%,greater than 99%, between about 60% to about 100%, between about 70% toabout 100%, between about 80% to about 100%, between about 90% to about100%, between about 95% to about 100%, or between about 99% to about100%, of an APPL attached to formula (i-a1).

In other embodiments, wherein the epoxide, thiirane, or aziridine offormula (i-x2) is chiral, the conjugation reaction is regioselective,and the reaction provides a chiral mixture of APPLs comprising moreAPPLs conjugated to a group of formula (i-a2) than formula (i-b2), e.g.,the reaction mixture comprises greater than 50%, greater than 60%,greater than 70%, greater than 80%, greater than 90%, greater than 95%,greater than 99%, between about 60% to about 100%, between about 70% toabout 100%, between about 80% to about 100%, between about 90% to about100%, between about 95% to about 100%, or between about 99% to about100%, of an APPL attached to formula (i-a2).

In certain embodiments, an APPL, and in particular, a compound ofFormulae (I), (II), (III), (IV), (V), or (VI), comprises at least oneinstance of a group of the formula (ii) attached thereto:

In certain embodiments of formula (ii), X is O. In certain embodimentsof formula (ii), X is S. In certain embodiments of formula (ii), X isNR^(X), wherein R^(X) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group. In certain embodiments of formula (ii), X isNR^(X), wherein R^(X) is hydrogen, optionally substituted alkyl, or anitrogen protecting group. In certain embodiments of formula (i), eachinstance of R′ is hydrogen.

In certain embodiments, an APPL, and in particular, a compound ofFormulae (I), (II), (III), (IV), (V), or (VI), comprises at least oneinstance of a group of the formula (ii) attached thereto:

As generally defined above, R^(L) is optionally substituted C₁₋₅₀ alkyl,optionally substituted C₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀alkynyl, optionally substituted C₁₋₅₀ heteroalkyl, optionallysubstituted C₂₋₅₀ heteroalkenyl, optionally substituted C₂₋₅₀heteroalkynyl, or a polymer. The group R^(L) seeks to encompasslipophilic, hydrophobic, and/or non-polar groups, but such terms shouldnot limit the scope of R^(L).

In certain embodiments, at least one instance of R^(L) is an optionallysubstituted C₁₋₅₀ alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₆₋₅₀alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₆₋₄₀alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₆₋₃₀alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₆₋₂₀alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₈₋₂₀alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₈alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₉alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₀alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₁alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₂alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₃alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₄alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₅alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₆alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₇alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₈alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₁₉alkyl. In certain embodiments, R^(L) is an optionallysubstituted C₂₀alkyl. In any of the above embodiments, the group R^(L)is an unsubstituted alkyl group.

In certain embodiments, at least one instance of R^(L) is anunsubstituted alkyl. Exemplary unsubstituted alkyl groups include, butare not limited to, —CH₃, —C₂H₅, —C₃H₇, —C₄H₉, —C₅H₁₁, —C₆H₁₃, —C₇H₁₅,—C₈H₁₇, —C₉H₁₉, —C₁₀H₂₁, —C₁₁H₂₃, —C₁₂H₂₅, —C₁₃H₂₇, —C₁₄H₂₉, —C₁₅H₃₁,—C₁₆H₃₃, —C₁₇H₃₅, —C₁₈H₃₇, —C₁₉H₃₉, —C₂₀H₄₁, —C₂₁H₄₃, —C₂₂H₄₅, —C₂₃H₄₇,—C₂₄H₄₉, and —C₂₅H₅₁.

In certain embodiments, at least one instance of R^(L) is a substitutedalkyl. For example, in certain embodiments, at least one instance ofR^(L) is an alkyl substituted with one or more fluorine substituents.Exemplary fluorinated alkyl groups include, but are not limited to:

In certain embodiments, at least one instance of R^(L) is an optionallysubstituted C₂₋₅₀ alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₆₋₅₀alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₆₋₄₀alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₆₋₃₀alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₆₋₂₀alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₈₋₂₀alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₈alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₉alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₀alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₁alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₂alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₃alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₄alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₅alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₆alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₇alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₈alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₁₉alkenyl. In certain embodiments, R^(L) is anoptionally substituted C₂₀alkenyl. In any of the above embodiments, thegroup R^(L) is an unsubstituted alkenyl group.

Exemplary unsubstituted alkenyl groups include, but are not limited to:

-   Myristoleic —(CH₂)₇CH═CH(CH₂)₃CH₃,-   Palmitoliec —(CH₂)₇CH═CH(CH₂)₅CH₃,-   Sapienic —(CH₂)₄CH═CH(CH₂)₈CH₃,-   Oleic —(CH₂)₇CH═CH(CH₂)₇CH₃,-   Linoleic —(CH₂)₇CH═CHCH₂CH═CH(CH₂)₄CH₃,-   α-Linolenic —(CH₂)₇CH═CHCH₂CH═CHCH₂CH═CHCH₂CH₃,-   Arachinodonic —(CH₂)₃CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₄CH₃,-   Eicosapentaenoic —(CH₂)₃CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH₃,-   Erucic —(CH₂)₁₁CH═CH(CH₂)₇CH₃, and-   Docosahexaenoic —(CH₂)₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═C    H—CH₂CH₃.

In embodiments, wherein R^(L) is defined as a C₆₋₅₀alkyl or C₆₋₅₀alkenylgroups, such groups are meant to encompass lipophilic groups (alsoreferred to as a “lipid tail”). Lipophilic groups comprise a group ofmolecules that include fats, waxes, oils, fatty acids, and the like.Lipid tails present in these lipid groups can be saturated andunsaturated, depending on whether or not the lipid tail comprises doublebonds. The lipid tail can also comprise different lengths, oftencategorized as medium (i.e., with tails between 7-12 carbons, e.g.,C₇₋₁₂ alkyl or C₇₋₁₂ alkenyl), long (i.e., with tails greater than 12carbons and up to 22 carbons, e.g., C₁₃₋₂₂ alkyl or C₁₃₋₂₂ alkenyl), orvery long (i.e., with tails greater than 22 carbons, e.g., C₂₃₋₃₀ alkylor C₂₃₋₃₀ alkenyl).

In certain embodiments, R^(L) is an optionally substituted C₂₋₅₀alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₆₋₅₀alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₆₋₄₀alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₆₋₃₀alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₆₋₂₀alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₈₋₂₀alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₈alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₉alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₀alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₁alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₂alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₃alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₄alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₅alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₆alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₇alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₈alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₁₉alkynyl. In certain embodiments, R^(L) is an optionally substitutedC₂₀alkynyl. In any of the above embodiments, the group R^(L) is anunsubstituted alkynyl group.

In certain embodiments, at least one instance of R^(L) is an optionallysubstituted heteroC₁₋₅₀ alkyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₆₋₅₀ alkyl. In certain embodiments, R^(L)is an optionally substituted heteroC₆₋₄₀ alkyl. In certain embodiments,R^(L) is an optionally substituted heteroC₆₋₃₀alkyl. In certainembodiments, R^(L) is an optionally substituted heteroC₆₋₂₀alkyl. Incertain embodiments, R^(L) is an optionally substitutedheteroC₁₀₋₂₀alkyl. In certain embodiments, R^(L) is an optionallysubstituted heteroC₈alkyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₉alkyl. In certain embodiments, R^(L) isan optionally substituted heteroC₁₀alkyl. In certain embodiments, R^(L)is an optionally substituted heteroC₁₁alkyl. In certain embodiments,R^(L) is an optionally substituted heteroC₁₂alkyl. In certainembodiments, R^(L) is an optionally substituted heteroC₁₃alkyl. Incertain embodiments, R^(L) is an optionally substituted heteroC₁₄alkyl.In certain embodiments, R^(L) is an optionally substitutedheteroC₁₈alkyl. In certain embodiments, R^(L) is an optionallysubstituted heteroC₁₆alkyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₁₇alkyl. In certain embodiments, R^(L) isan optionally substituted heteroC₁₈alkyl. In certain embodiments, R^(L)is an optionally substituted heteroC₁₉alkyl. In certain embodiments,R^(L) is an optionally substituted heteroC₂₀alkyl. In any of the aboveembodiments, the group R^(L) is an unsubstituted heteroalkyl group.

Exemplary unsubstituted heteroalkyl groups include, but are not limitedto,

In certain embodiments, at least one instance of R^(L) is an optionallysubstituted heteroC₂₋₅₀alkenyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₆₋₅₀alkenyl. In certain embodiments, R^(L)is an optionally substituted heteroC₆₋₄₀alkenyl. In certain embodiments,R^(L) is an optionally substituted heteroC₆₋₃₀alkenyl. In certainembodiments, R^(L) is an optionally substituted heteroC₆₋₂₀alkenyl. Incertain embodiments, R^(L) is an optionally substitutedheteroC₈₋₂₀alkenyl. In certain embodiments, R^(L) is an optionallysubstituted heteroC₈alkenyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₉alkenyl. In certain embodiments, R^(L) isan optionally substituted heteroC₁₀alkenyl. In certain embodiments,R^(L) is an optionally substituted heteroC₁₁alkenyl. In certainembodiments, R^(L) is an optionally substituted heteroC₁₂alkenyl. Incertain embodiments, R^(L) is an optionally substitutedheteroC₁₃alkenyl. In certain embodiments, R^(L) is an optionallysubstituted heteroC₁₄alkenyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₁₅alkenyl. In certain embodiments, R^(L)is an optionally substituted heteroC₁₆alkenyl. In certain embodiments,R^(L) is an optionally substituted heteroC₁₇alkenyl. In certainembodiments, R^(L) is an optionally substituted heteroC₁₈alkenyl. Incertain embodiments, R^(L) is an optionally substitutedheteroC₁₉alkenyl. In certain embodiments, R^(L) is an optionallysubstituted heteroC₂₀alkenyl. In any of the above embodiments, the groupR^(L) is an unsubstituted heteroalkenyl group.

In certain embodiments, R^(L) is an optionally substitutedheteroC₂₋₅₀alkynyl. In certain embodiments, R^(L) is an optionallysubstituted heteroC₆₋₅₀alkynyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₆₋₄₀alkynyl. In certain embodiments, R^(L)is an optionally substituted heteroC₆₋₃₀alkynyl. In certain embodiments,R^(L) is an optionally substituted heteroC₆₋₂₀alkynyl. In certainembodiments, R^(L) is an optionally substituted heteroC₈₋₂₀alkynyl. Incertain embodiments, R^(L) is an optionally substituted heteroC₈alkynyl.In certain embodiments, R^(L) is an optionally substitutedheteroC₉alkynyl. In certain embodiments, R^(L) is an optionallysubstituted heteroC₁₀alkynyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₁₁alkynyl. In certain embodiments, R^(L)is an optionally substituted heteroC₁₂alkynyl. In certain embodiments,R^(L) is an optionally substituted heteroC₁₃alkynyl. In certainembodiments, R^(L) is an optionally substituted heteroC₁₄alkynyl. Incertain embodiments, R^(L) is an optionally substitutedheteroC₁₅alkynyl. In certain embodiments, R^(L) is an optionallysubstituted heteroC₁₆alkynyl. In certain embodiments, R^(L) is anoptionally substituted heteroC₁₇alkynyl. In certain embodiments, R^(L)is an optionally substituted heteroC₁₈alkynyl. In certain embodiments,R^(L) is an optionally substituted heteroC₁₉alkynyl. In certainembodiments, R^(L) is an optionally substituted heteroC₂₀alkynyl. In anyof the above embodiments, the group R^(L) is an unsubstitutedheteroalkynyl group.

In certain embodiments, at least one instance of R^(L) is a polymer. Asused herein, a “polymer” refers to a compound comprised of at least 3(e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeatingcovalently bound structural units. The polymer is in certain embodimentsbiocompatible (i.e., non-toxic). Exemplary polymers include, but are notlimited to, cellulose polymers (e.g., hydroxyethylcellulose,ethylcellulose, carboxymethylcellulose, methylc cellulose,hydroxypropylmethylcellulose (HPMC)), dextran polymers, polymaleic acidpolymers, poly(acrylic acid) polymers, poly(vinylalcohol) polymers,polyvinylpyrrolidone (PVP) polymers, and polyethyleneglycol (PEG)polymers, and combinations thereof.

Additional Methods of Preparation

As described herein, in order to provide compounds of the presentinvention, an APPL precursor is treated with one or more conjugatingreagents, e.g., selected from an epoxide, thiirane, or aziridine offormula (i-x), an α,β-unsaturated ester, thioester, or amide of formula(ii-x), or an an aldehyde of formula (iii-x), to provide the APPL.

For example, in one aspect, provided is a method of preparing an APPLfunctionalized with a group of formula (i) comprising heating theprecursor in an organic solvent (e.g., EtOH) with one or moreconjugating reagents of formula (i-x) to provide the desired APPL. Incertain embodiments, the mixture is heated between about 100 to about200° C., inclusive, e.g., about 150° C.

In another aspect, provided is a method of preparing an APPLfunctionalized with a group of formula (ii) comprising heating theprecursor in an organic solvent (e.g., EtOH) with one or moreconjugating reagents of formula (ii-x) to provide the desired APPL. Incertain embodiments, the mixture is heated between about 50 to about100° C., inclusive, e.g., about 90° C.

In another aspect, provided is a method of preparing an APPLfunctionalized with a group of formula (iii) comprising mixing theprecursor in an organic solvent (e.g., THF) with one or more conjugatingreagents of formula (iii-x) and a reducing agent (e.g., NaBH(OAc)₃) toprovide the desired APPL. In certain embodiments, the temperature of thereaction mixture is room temperature mixture.

In certain embodiments, wherein only one conjugating reagent is used,each instance of R^(L) is the same in the APPL. For example, in certainembodiments, each instance of R^(L) is the same wherein R^(L) is anoptionally substituted alkyl. In certain embodiments, each instance ofR^(L) is the same wherein R^(L) is an unsubstituted alkyl. In certainembodiments, each instance of R^(L) is the same wherein R^(L) isselected from the group consisting of —CH₃, —C₂H₅, —C₃H₇, —C₄H₉, —C₅H₁₁,—C₆H₁₃, —C₇H₁₅, —C₈H₁₇, —C₉H₁₉, —C₁₀H₂₁, —C₁₁H₂₃, —C₁₂H₂₅, —C₁₃H₂₇,—C₁₄H₂₉, —C₁₅H₃₁, —C₁₆H₃₃, —C₁₇H₃₅, —C₁₈H₃₇, —C₁₉H₃₉, and —C₂₀H₄₁. Incertain embodiments, each instance of R^(L) is the same wherein R^(L) isan n-alkyl group selected from —C₈H₁₇, —C₉H₁₉, —C₁₀H₂₁, —C₁₁H₂₃,—C₁₂H₂₅, —C₁₃H₂₇, —C₁₄H₂₉, —C₁₅H₃₁, and —C₁₆H₃₃.

Alternatively, in certain embodiments, wherein more than one conjugatingreagent is used in the conjugation reaction (e.g., two, three, four,five, six, seven, eight, nine, or ten different conjugating reagents),the APPL may comprise two or more (e.g., two, three, four, five, six,seven, eight, nine, or ten) different groups of the formula (i), (ii),and/or (iii) attached thereto.

For example, in certain embodiments, two different epoxides are used inthe conjugation reaction. In this instance, in certain embodiments, theAPPL comprises two different R^(L) groups. For example, in certainembodiments, the APPL comprises a mixture of two different R^(L) groups,wherein the first R^(L) group is an optionally substituted alkyl, andthe second R^(L) group is a polymer.

As would be appreciated by one of skill in this art, the degree ofconjugation may be controlled by the reaction conditions (e.g.,temperature, starting materials, concentration, solvent, etc.) used inthe synthesis. The synthesized APPL may be purified by any techniqueknown in the art including, but not limited to, precipitation,crystallization, chromatography, distillation, etc.

In certain embodiments, the APPL is isolated as a salt. For example, incertain embodiments, the APPL is reacted with an acid (e.g., an organicacid or inorganic acid) to form the corresponding salt. In otherembodiments, tertiary amines are alkylated to form a quaternary ammoniumsalt of the APPL. The tertiary amines may be alkylated with anyalkylating agent, for example, alkyl halides such as methyl iodide maybe used to from the quaternary amino groups. The anion associated withthe quaternary amine may be any organic or inorganic anion. In certainembodiments, the anion is a pharmaceutically acceptable anion.

The invention also provides libraries of APPLs prepared by the inventivemethods. For example, in certain embodiments, provided is a method ofscreening a compound library, the method comprising providing aplurality of different APPLs, or salts thereof; and performing at leastone assay with the compound library to determine the presense or absenceof a desired property. These APPLs may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the APPLs are screenedfor their ability to transfect polynucleotides or other agents (e.g.,proteins, peptides, small molecules) into the cell. For example, in oneembodiment, provided is a method of screening a compound library, themethod comprising providing a plurality of two or more different APPLsand screening the compound library for a desired property.

In one embodiment, a library of different APPLs is prepared in parallel.A different precursor and/or conjugating reagent is added to each vialin a set of vials or to each well of a multi-well plate used to preparethe library. The array of reaction mixtures is incubated at atemperature and length of time sufficient to allow formation of theAPPL. The APPL may then be isolated and purified using techniques knownin the art. The APPL may then be screened using high-throughputtechniques to identify APPLs with a desired property, e.g., wherein thedesired property is solubility in water, solubility at different pH,ability to bind polynucleotides, ability to bind heparin, ability tobind small molecules, ability to bind protein, ability to formmicroparticles, ability to increase tranfection efficiency, ability tosupport cell growth, ability to support cell attachment, ability tosupport tissue growth, and/or intracellular delivery of the APPL and/oran agent complexed or attached thereto to aid in bioprocessing, e.g.,for the purpose of manufacturing proteins. In certain embodiments theAPPLs may be screened for properties or characteristics useful ascoatings, additives, materials, and excipients in biotechnology andbiomedical applications such as the coating of medical devices orimplants with films or multilayer films, as non-biofouling agents,micropatterning agents, and cellular encapsulation agents. In certainembodiments the APPL may be screened for properties or characteristicsuseful in gene therapy (e.g., the ability to bind polynucleotides and/orincrease in transfection efficiency), bioprocessing (e.g., aiding in theintracellular manufacturing of proteins), or the administration and/ordelivery of a therapeutic agent (e.g., polynucleotide, small molecule,antigen, drug, protein, peptide, etc.) to a subject, tissue, organ, orcell.

Exemplary Compounds of the Present Invention

Certain compounds of the present invention are specifically contemplatedherein. For example, compounds comprising unsubstituted n-alkyl R^(L)groups containing 8, 9, 10, 11, 12, 13, and 14 carbon atoms arespecifically contemplated. In certain embodiments R¹ of such compoundsis an amino acid side chain as defined in Table 1 of the Examples.

Exemplary amino acid, peptide, and polypeptide compounds of Formula (I)include, but are not limited to:

and salts thereof.

Exemplary cyclized compounds of Formula (II), include, but are notlimited to:

and salts thereof.

Exemplary cyclic dipeptide and cyclic polypeptide compounds of Formula(III) include, but are not limited to:

and, in particular, cyclic-KK and polycyclic lysine APPLs of theformula:

and salts thereof.

Compositions

The present invention contemplates an APPL as a component of acomposition. For example, in certain embodiments, provided is acomposition comprising an APPL, or salt thereof, and an excipient,wherein the APPL is an amino acid, a linear or cyclic peptide, or alinear or cyclic polypeptide, or structural isomer thereof, and whereinan amino or amide group of the APPL is conjugated to a group of formula(i), (ii), or (iii). In certain embodiments, the group of formula (i),(ii), or (iii) is attached to an amino group present on the APPLscaffold.

Compositions, as described herein, comprising an APPL and an excipientof some sort may be useful in a variety of medical and non-medicalapplications. For example, pharmaceutical compositions comprising anAPPL and an excipient may be useful in the delivery of an effectiveamount of an agent to a subject in need thereof. Nutraceuticalcompositions comprising an APPL and an excipient may be useful in thedelivery of an effective amount of a nutraceutical, e.g., a dietarysupplement, to a subject in need thereof. Cosmetic compositionscomprising an APPL and an excipient may be formulated as a cream,ointment, balm, paste, film, or liquid, etc., and may be useful in theapplication of make-up, hair products, and materials useful for personalhygiene, etc. Compositions comprising an APPL and an excipient may beuseful for non-medical applications, e.g., such as an emulsion oremulsifier, useful, for example, as a food component, for extinguishingfires, for disinfecting surfaces, for oil cleanup, etc.

Peptides play significant roles in endogenous cellular signaling andtrafficking pathways, and offer tremendous potential in leveraging suchinteractions to enhance the delivery efficiency of systems whichincorporate peptide moieties. Thus, compositions comprising an APPL andan excipient may further be useful in bioprocessing, such as a cell'sbioprocessing of a commercially useful chemical or fuel. For example,intracellular delivery of the APPL or an agent complexed thereto may beuseful in bioprocessing by maintaining the cell's health and/or growth,e.g., in the manufacturing of proteins.

The composition may comprise one type of APPL but may also comprise anynumber of different types of APPLs, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more different types of APPLs.

In certain embodiments, the composition further comprises an agent, asdescribed herein. For example, in certain embodiments, the agent is asmall molecule, organometallic compound, nucleic acid, protein, peptide,polynucleotide, metal, targeting agent, an isotopically labeled chemicalcompound, drug, vaccine, immunological agent, or an agent useful inbioprocessing. In certain embodiments, the agent is a polynucleotide. Incertain embodiments, the polynucleotide is DNA or RNA. In certainembodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisenseRNA. In certain embodiments, the polynucleotide and the one or moreAPPLs are not covalently attached.

In certain embodiments, the one or more APPLs are in the form of aparticle. In certain embodiments, the particle is a nanoparticle ormicroparticle. In certain embodiments, the one or more APPLs are in theform of liposomes or micelles. It is understood that, in certainembodiments, these APPLs self-assemble to provide a particle, micelle,or liposome. In certain embodiments, the particle, micelle, or liposomeencapsulates an agent. The agent to be delivered by the particle,micelle, or liposome may be in the form of a gas, liquid, or solid. TheAPPLs may be combined with polymers (synthetic or natural), surfactants,cholesterol, carbohydrates, proteins, lipids etc. to form the particles.These particles may be further combined with an excipient to form thecomposition.

“Excipients” include any and all solvents, diluents or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. General considerations in formulation and/or manufacture can befound, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21st Edition(Lippincott Williams & Wilkins, 2005).

Exemplary excipients include, but are not limited to, any non-toxic,inert solid, semi-solid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. Some examples ofmaterials which can serve as excipients include, but are not limited to,sugars such as lactose, glucose, and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose, and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil;safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycolssuch as propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; detergents such as Tween 80; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator. As wouldbe appreciated by one of skill in this art, the excipients may be chosenbased on what the composition is useful for. For example, with apharmaceutical composition or cosmetic composition, the choice of theexcipient will depend on the route of administration, the agent beingdelivered, time course of delivery of the agent, etc., and can beadministered to humans and/or to animals, orally, rectally,parenterally, intracisternally, intravaginally, intranasally,intraperitoneally, topically (as by powders, creams, ointments, ordrops), bucally, or as an oral or nasal spray.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, etc., and combinationsthereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g. acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g.bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]),long chain amino acid derivatives, high molecular weight alcohols (e.g.stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate,ethylene glycol distearate, glyceryl monostearate, and propylene glycolmonostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene,polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer),carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium,powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acidesters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20],polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate[Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate[Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitanmonooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylenemonostearate [Myrj 45], polyoxyethylene hydrogenated castor oil,polyethoxylated castor oil, polyoxymethylene stearate, and Solutol),sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether[Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate,triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate,oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68,Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride,benzalkonium chloride, docusate sodium, etc. and/or combinationsthereof.

Exemplary binding agents include starch (e.g. cornstarch and starchpaste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghattigum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, microcrystalline cellulose, celluloseacetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum),and larch arabogalactan), alginates, polyethylene oxide, polyethyleneglycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes,water, alcohol, etc., and/or combinations thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, etc., and combinations thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, andcombinations thereof.

Additionally, the composition may comprise a phospholipid. Exemplaryphospholipids include, but are not limited to,disteroylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine(DMPC), Dipalmitoylphosphatidylcholine (DPPC), anddioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-Dilauroyl-sn-Glycero-3-Phosphocholine (dilauroylphosphatidylcholine,DLPC), 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine(dimyristoylphosphatidylcholine, DMPC),1,2-Dipentadecanoyl-sn-Glycero-3-Phosphocholine(dipentadecanoylphosphatidylcholine, DPDPC),1,2-dipalmitoyl-sn-Glycero-3-Phosphocholine(dipalmitoylphosphatidylcholine, DPPC),1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine(1-myristoyl-2-palmitoylphosphatidylcholine, MPPC),1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG), and1,2-Dimyristoyl-3-Trimethylammonium-propane.

Additionally, the composition may further comprise a polymer. Exemplarypolymers contemplated herein include, but are not limited to, cellulosicpolymers and copolymers, for example, cellulose ethers such asmethylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC),methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC),carboxymethyl cellulose (CMC) and its various salts, including, e.g.,the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and itsvarious salts, carboxymethylhydroxyethylcellulose (CMHEC) and itsvarious salts, other polysaccharides and polysaccharide derivatives suchas starch, dextran, dextran derivatives, chitosan, and alginic acid andits various salts, carageenan, varoius gums, including xanthan gum, guargum, gum arabic, gum karaya, gum ghatti, konjac and gum tragacanth,glycosaminoglycans and proteoglycans such as hyaluronic acid and itssalts, proteins such as gelatin, collagen, albumin, and fibrin, otherpolymers, for example, polyhydroxyacids such as polylactide,polyglycolide, polyl(lactide-co-glycolide) andpoly(.epsilon.-caprolactone-co-glycolide)-, carboxyvinyl polymers andtheir salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylicacid and its salts, polyacrylamide, polyacilic acid/acrylamidecopolymer, polyalkylene oxides such as polyethylene oxide, polypropyleneoxide, poly(ethylene oxide-propylene oxide), and a Pluronic polymer,polyoxyethylene (polyethylene glycol), polyanhydrides, polyvinylalchol,polyethyleneamine and polypyrridine, polyethylene glycol (PEG) polymers,such as PEGylated lipids (e.g., PEG-stearate,1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-1000],1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-2000], and1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-5000]), copolymers and salts thereof.

Additionally, the composition may further comprise an emulsifying agent.Exemplary emulsifying agents include, but are not limited to, apolyethylene glycol (PEG), a polypropylene glycol, a polyvinyl alcohol,a poly-N-vinyl pyrrolidone and copolymers thereof, poloxamer nonionicsurfactants, neutral water-soluble polysaccharides (e.g., dextran,Ficoll, celluloses), non-cationic poly(meth)acrylates, non-cationicpolyacrylates, such as poly(meth)acrylic acid, and esters amide andhydroxyalkyl amides thereof, natural emulsifiers (e.g. acacia, agar,alginic acid, sodium alginate, tragacanth, chondrux, cholesterol,xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax,and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] andVeegum [magnesium aluminum silicate]), long chain amino acidderivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetylalcohol, oleyl alcohol, triacetin monostearate, ethylene glycoldistearate, glyceryl monostearate, and propylene glycol monostearate,polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylicacid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan,cellulosic derivatives (e.g. carboxymethylcellulose sodium, powderedcellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acidesters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20],polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate[Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate[Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitanmonooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylenemonostearate [Myrj 45], polyoxyethylene hydrogenated castor oil,polyethoxylated castor oil, polyoxymethylene stearate, and Solutol),sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether[Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate,triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate,oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68,Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride,benzalkonium chloride, docusate sodium, etc. and/or combinationsthereof. In certain embodiments, the emulsifying agent is cholesterol.

Additionally, the composition may further comprise an apolipoprotein.Previous studies have reported that Apolipoprotein E (ApoE) was able toenhance cell uptake and gene silencing for a certain type of materials.See, e.g., Akinc, A., et al., Targeted delivery of RNAi therapeuticswith endogenous and exogenous ligand-based mechanisms. Mol Ther. 18(7):p. 1357-64. In certain embodiments, the apolipoprotein is ApoA, ApoB,ApoC, ApoE, or ApoH, or an isoform thereof.

Liquid compositions include emulsions, microemulsions, solutions,suspensions, syrups, and elixirs. In addition to the APPL, the liquidcomposition may contain inert diluents commonly used in the art such as,for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable compositions, for example, injectable aqueous or oleaginoussuspensions may be formulated according to the known art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a injectable solution, suspension, oremulsion in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol. Among the acceptable vehiclesand solvents for pharmaceutical or cosmetic compositions that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. Any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Incertain embodiments, the particles are suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween80. The injectable composition can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration may be in the form ofsuppositories which can be prepared by mixing the particles withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the particles.

Solid compositions include capsules, tablets, pills, powders, andgranules. In such solid compositions, the particles are mixed with atleast one excipient and/or a) fillers or extenders such as starches,lactose, sucrose, glucose, mannitol, and silicic acid, b) binders suchas, for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets, and pills, the dosage form may also comprise buffering agents.Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Tablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings and other coatings wellknown in the pharmaceutical formulating art. They may optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Compositions for topical or transdermal administration includeointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants, or patches. The APPL is admixed with an excipient and anyneeded preservatives or buffers as may be required. Ophthalmicformulation, ear drops, and eye drops are also contemplated as beingwithin the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theAPPL, excipients such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc, and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to the APPL, excipients suchas lactose, talc, silicic acid, aluminum hydroxide, calcium silicates,and polyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants such aschlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

Agents

Agents to be delivered by the systems described herein may betherapeutic, diagnostic, or prophylactic agents. Any chemical compoundto be administered to a subject may be delivered using the complexes,picoparticles, nanoparticles, microparticles, micelles, or liposomes,described herein. The agent may be an organic molecule (e.g., atherapeutic agent, a drug), inorganic molecule, nucleic acid, protein,amino acid, peptide, polypeptide, polynucleotide, targeting agent,isotopically labeled organic or inorganic molecule, vaccine,immunological agent, etc.

In certain embodiments, the agents are organic molecules withpharmaceutical activity, e.g., a drug. In certain embodiments, the drugis an antibiotic, anti-viral agent, anesthetic, steroidal agent,anti-inflammatory agent, anti-neoplastic agent, anti-cancer agent,antigen, vaccine, antibody, decongestant, antihypertensive, sedative,birth control agent, progestational agent, anti-cholinergic, analgesic,anti-depressant, anti-psychotic, β-adrenergic blocking agent, diuretic,cardiovascular active agent, vasoactive agent, non-steroidalanti-inflammatory agent, nutritional agent, etc.

In certain embodiments of the present invention, the agent to bedelivered may be a mixture of agents.

Diagnostic agents include gases; metals; commercially available imagingagents used in positron emissions tomography (PET), computer assistedtomography (CAT), single photon emission computerized tomography, x-ray,fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI includegadolinium chelates, as well as iron, magnesium, manganese, copper, andchromium. Examples of materials useful for CAT and x-ray imaging includeiodine-based materials.

Therapeutic and prophylactic agents include, but are not limited to,antibiotics, nutritional supplements, and vaccines. Vaccines maycomprise isolated proteins or peptides, inactivated organisms andviruses, dead organisms and viruses, genetically altered organisms orviruses, and cell extracts. Therapeutic and prophylactic agents may becombined with interleukins, interferon, cytokines, and adjuvants such ascholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents includeantigens of such bacterial organisms as Streptococcus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens ofsuch viruses as smallpox, influenza A and B, respiratory syncytialvirus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike; antigens of fungal, protozoan, and parasitic organisms such asCryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosomamansoni, and the like. These antigens may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, carbohydrates, orcombinations thereof.

Targeting Agents

Since it is often desirable to target a particular cell, collection ofcells, or tissue, an APPL, and the complexes, liposomes, micelles,microparticles, picoparticles and nanoparticles, prepared therefrom, maybe modified to include targeting agents or targeting regions. Forexample, the APPL scaffold may include a targeting region. A variety ofagents or regions that target particular cells are known in the art.See, e.g., Cotten et al., Methods Enzym. 217:618, 1993. The targetingagents may be included throughout the particle or may be only on thesurface. The targeting agent may be a protein, peptide, carbohydrate,glycoprotein, lipid, small molecule, nucleic acids, etc. The targetingagent may be used to target specific cells or tissues or may be used topromote endocytosis or phagocytosis of the particle. Examples oftargeting agents include, but are not limited to, antibodies, fragmentsof antibodies, low-density lipoproteins (LDLs), transferrin,asialycoproteins, gp120 envelope protein of the human immunodeficiencyvirus (HIV), carbohydrates, receptor ligands, sialic acid, aptamers,etc. If the targeting agent is included throughout the particle, thetargeting agent may be included in the mixture that is used to form theparticles. If the targeting agent is only on the surface, the targetingagent may be associated with (i.e., by covalent, hydrophobic, hydrogenbonding, van der Waals, or other interactions) the formed particlesusing standard chemical techniques.

Polynucleotide Complexes

The present invention contemplates APPLs are particularly useful in theadministration of polynucleotides. For example, APPLs comprise secondaryor tertiary amines, and, although these amines are hindered, they areavailable to non-covalently interact with a polynucleotide (e.g., DNA,RNA, synthetic analogs of DNA and/or RNA, DNA/RNA hydrids, etc.).Polynucleotides or derivatives thereof are contacted with an APPL underconditions suitable to form a polynucleotide/APPL non-covalent complex.The interaction of the APPL with the polynucleotide is thought to atleast partially prevent the degradation of the polynucleotide. Byneutralizing the charge on the backbone of the polynucleotide, theneutral or slightly-positively-charged complex is also able to moreeasily pass through the hydrophobic membranes (e.g., cytoplasmic,lysosomal, endosomal, nuclear) of the cell. In certain embodiments, thecomplex is slightly positively charged. In certain embodiments, thecomplex has a positive ζ-potential. In certain embodiments theζ-potential is between 0 and +30.

In one aspect, provided is a method of delivering a polynucleotide to abiological cell, comprising providing a composition comprising an APPL,or salt thereof, and a polynucleotide; and exposing the composition tothe biological cell under conditions sufficient to facilitate deliveryof the polynucleotide into the interior of the biological cell; whereinthe APPL is an amino acid, a linear or cyclic peptide, or a linear orcyclic polypeptide, or structural isomer thereof, wherein an amino oramide group of the APPL is conjugated to a group of formula (i), (ii),or (iii). In certain embodiments, the method is an in vivo method. Incertain embodiments, the method is an in vitro method.

An APPL may be at least partially provided as a salt (e.g., isprotonated) so as to form a complex with the negatively chargedpolynucleotide. In certain embodiments, the polynucleotide/APPL complexform particles that are useful in the delivery of polynucleotides tocells. In certain embodiments, more than one APPL may be associated witha polynucleotide molecule. For example, the complex may include 1-100APPLs, 1-1000 APPLs, 10-1000 APPLs, or 100-10,000 APPLs associated witha polynucleotide molecule.

Increasing nitrogen:phosphate ratios have been shown to positivelyinfluence delivery of genetic material by increasing nucleic acidbinding and negatively influence delivery by increasing toxicity. See,e.g., Incani et al., Soft Matter (2010) 6:2124-2138. In certainembodiments, the nitrogen:phosphate ratio (i.e., the ratio between theamino groups present in the APPL, and the phosphate groups present inthe polynucleotide) is between about 10:1 to about 50:1, inclusive. Incertain embodiments, the nitrogen phosphate ratio is between about 10:1to about 45:1, between about 15:1 to about 45:1, or between about 20:1to about 40:1, inclusive. In certain embodiments, theAPPL:polynucleotide mass ratio is between about 10:1 to about 20:1,inclusive. In certain embodiments, the APPL:polynucleotide mass ratio isabout 15:1. In certain embodiments, the APPL:polynucleotide molar ratiois between about 10:1 to about 400:1, inclusive. In certain embodiments,the APPL:polynucleotide molar ratio is between about 10:1 to about350:1, between about 15:1 to about 300:1, or between about 20:1 to about250:1, inclusive.

In certain embodiments, the complex may form a particle. In certainembodiments, the diameter of the particles ranges from 10-500micrometers. In certain embodiments, the diameter of the particlesranges from 10-1200 micrometers. In certain embodiments, the diameter ofthe particles ranges from 50-150 micrometers. In certain embodiments,the diameter of the particles ranges from 10-500 nm, in certainembodiments the diameter of the particles ranges from 10-1200 nm, and incertain embodiments from 50-150 nm. The particles may be associated witha targeting agent as described below. In certain embodiments, thediameter of the particles ranges from 10-500 pm, in certain embodimentsthe diameter of the particles ranges from 10-1200 pm, and in certainembodiments from 50-150 pm. The particles may be associated with atargeting agent as described below. The film architecture is preciselydesigned and can be controlled to 1 nm precision with a range from 1 to150000 nm and with a definite knowledge of its molecular composition.

The polynucleotide may be complexed, encapsulated by an APPL, orincluded in a composition comprising an APPL. The polynucleotide may beany nucleic acid including, but not limited to, RNA and DNA. In certainembodiments, the polynucleotide is DNA. In certain embodiments, thepolynucleotide is RNA. In certain embodiments, upon delivery of the RNAinto a cell, the RNA is able to interfere with the expression of aspecific gene in the biological cell.

In certain embodiments, the polynucleotide is an RNA that carries outRNA interference (RNAi). The phenomenon of RNAi is discussed in greaterdetail, for example, in the following references: Elbashir et al., 2001,Genes Dev., 15:188; Fire et al., 1998, Nature, 391:806; Tabara et al.,1999, Cell, 99:123; Hammond et al., Nature, 2000, 404:293; Zamore etal., 2000, Cell, 101:25; Chakraborty, 2007, Curr. Drug Targets, 8:469;and Morris and Rossi, 2006, Gene Ther., 13:553. In certain embodiments,the polynucleotide is a dsRNA (double-stranded RNA). In certainembodiments, the polynucleotide is an siRNA (short interfering RNA). Incertain embodiments, the polynucleotide is an shRNA (short hairpin RNA).In certain embodiments, the polynucleotide is an miRNA (micro RNA).Micro RNAs (miRNAs) are genomically encoded non-coding RNAs of about21-23 nucleotides in length that help regulate gene expression,particularly during development. See, e.g., Bartel, 2004, Cell, 116:281;Novina and Sharp, 2004, Nature, 430:161; and U.S. Patent Publication2005/0059005; also reviewed in Wang and Li, 2007, Front. Biosci.,12:3975; and Zhao, 2007, Trends Biochem. Sci., 32:189. In certainembodiments, the polynucleotide is an antisense RNA.

In certain embodiments, the polynucleotide may be provided as anantisense agent or RNA interference (RNAi). See, e.g., Fire et al.,Nature 391:806-811, 1998. Antisense therapy is meant to include, e.g.,administration or in situ provision of single- or double-strandedoligonucleotides or their derivatives which specifically hybridize,e.g., bind, under cellular conditions, with cellular mRNA and/or genomicDNA, or mutants thereof, so as to inhibit expression of the encodedprotein, e.g., by inhibiting transcription and/or translation. See,e.g., Crooke “Molecular mechanisms of action of antisense drugs”Biochim. Biophys. Acta 1489(1):31-44, 1999; Crooke “Evaluating themechanism of action of antiproliferative antisense drugs” AntisenseNucleic Acid Drug Dev. 10(2):123-126, discussion 127, 2000; Methods inEnzymology volumes 313-314, 1999. The binding may be by conventionalbase pair complementarity, or, for example, in the case of binding toDNA duplexes, through specific interactions in the major groove of thedouble helix (i.e., triple helix formation). See, e.g., Chan et al., J.Mol. Med. 75(4):267-282, 1997.

In some embodiments, dsRNA, siRNA, shRNA, miRNA, antisense RNA, and/orRNAi can be designed and/or predicted using one or more of a largenumber of available algorithms. To give but a few examples, thefollowing resources can be utilized to design and/or predictpolynucleotides: algorithms found at Alnylum Online, Dharmacon Online,OligoEngine Online, Molecula Online, Ambion Online, BioPredsi Online,RNAi Web Online, Chang Bioscience Online, Invitrogen Online, LentiWebOnline GenScript Online, Protocol Online; Reynolds et al., 2004, Nat.Biotechnol., 22:326; Naito et al., 2006, Nucleic Acids Res., 34:W448; Liet al., 2007, RNA, 13:1765; Yiu et al., 2005, Bioinformatics, 21:144;and Jia et al., 2006, BMC Bioinformatics, 7: 271.

The polynucleotides may be of any size or sequence, and they may besingle- or double-stranded. In certain embodiments, the polynucleotideis greater than 100 base pairs long. In certain embodiments, thepolynucleotide is greater than 1000 base pairs long and may be greaterthan 10,000 base pairs long. The polynucleotide is optionally purifiedand substantially pure. In certain embodiments, the polynucleotide isgreater than 50% pure, in certain embodiments greater than 75% pure, andin certain embodiments greater than 95% pure. The polynucleotide may beprovided by any means known in the art. In certain embodiments, thepolynucleotide has been engineered using recombinant techniques. See,e.g., Ausubel et al., Current Protocols in Molecular Biology (John Wiley& Sons, Inc., New York, 1999); Molecular Cloning: A Laboratory Manual,2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring HarborLaboratory Press: 1989). The polynucleotide may also be obtained fromnatural sources and purified from contaminating components foundnormally in nature. The polynucleotide may also be chemicallysynthesized in a laboratory. In certain embodiments, the polynucleotideis synthesized using standard solid phase chemistry.

The polynucleotide may be modified by chemical or biological means. Incertain embodiments, these modifications lead to increased stability ofthe polynucleotide. Modifications include methylation, phosphorylation,end-capping, etc.

Derivatives of polynucleotides may also be used in the presentinvention. These derivatives include modifications in the bases, sugars,and/or phosphate linkages of the polynucleotide. Modified bases include,but are not limited to, those found in the following nucleoside analogs:2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine. Modified sugarsinclude, but are not limited to, 2′-fluororibose, ribose,2′-deoxyribose, 3′-azido-2′,3′-dideoxyribose, 2′,3′-dideoxyribose,arabinose (the 2′-epimer of ribose), acyclic sugars, and hexoses. Thenucleosides may be strung together by linkages other than thephosphodiester linkage found in naturally occurring DNA and RNA.Modified linkages include, but are not limited to, phosphorothioate and5′-N-phosphoramidite linkages. Combinations of the various modificationsmay be used in a single polynucleotide. These modified polynucleotidesmay be provided by any means known in the art; however, as will beappreciated by those of skill in this art, the modified polynucleotidesmay be prepared using synthetic chemistry in vitro.

The polynucleotides to be delivered may be in any form. For example, thepolynucleotide may be a circular plasmid, a linearized plasmid, acosmid, a viral genome, a modified viral genome, an artificialchromosome, etc.

The polynucleotide may be of any sequence. In certain embodiments, thepolynucleotide encodes a protein or peptide. The encoded proteins may beenzymes, structural proteins, receptors, soluble receptors, ionchannels, pharmaceutically active proteins, cytokines, interleukins,antibodies, antibody fragments, antigens, coagulation factors, albumin,growth factors, hormones, insulin, etc. The polynucleotide may alsocomprise regulatory regions to control the expression of a gene. Theseregulatory regions may include, but are not limited to, promoters,enhancer elements, repressor elements, TATA box, ribosomal bindingsites, stop site for transcription, etc. In certain embodiments, thepolynucleotide is not intended to encode a protein. For example, thepolynucleotide may be used to fix an error in the genome of the cellbeing transfected.

In certain embodiments, the polynucleotide to be delivered comprises asequence encoding an antigenic peptide or protein. Nanoparticlescontaining these polynucleotides can be delivered to an individual toinduce an immunologic response sufficient to decrease the chance of asubsequent infection and/or lessen the symptoms associated with such aninfection. The polynucleotide of these vaccines may be combined withinterleukins, interferon, cytokines, and adjuvants such as choleratoxin, alum, Freund's adjuvant, etc. A large number of adjuvantcompounds are known; a useful compendium of many such compounds isprepared by the National Institutes of Health. See, e.g., Allison Dev.Biol. Stand. 92:3-11, 1998; Unkeless et al., Annu. Rev. Immunol.6:251-281, 1998; and Phillips et al., Vaccine 10:151-158, 1992.

The antigenic protein or peptides encoded by the polynucleotide may bederived from such bacterial organisms as Streptococcus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; from suchviruses as smallpox, influenza A and B, respiratory syncytial virus,parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2,cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus,papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, hepatitis A, B, C, D, and E virus, and the like; and from suchfungal, protozoan, and parasitic organisms such as Cryptococcusneoformans, Histoplasma capsulatum, Candida albicans, Candidatropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi,Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis,Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica,Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and thelike.

Particles

The present invention also contemplates APPLs useful as a deliverydevice. APPLs have several properties that make them particularlysuitable for delivery, including: 1) the ability of an APPL to complexand “protect” labile agents; 2) the ability to buffer the pH in theendosome; 3) the ability to act as a “proton sponge” and causeendosomolysis; and 4) the ability to neutralize the charge on negativelycharged agents.

In certain embodiments, an APPL is used to form particles containing theagent to be delivered. An APPL may be used to encapsulate agentsincluding, but not limited to, organic molecules (e.g., cholesterol,drugs), inorganic molecules, nucleic acids, proteins, peptides,polynucleotides, targeting agents, isotopically labeled organic orinorganic molecules, vaccines, immunological agents, etc. Otherexemplary agents are described in greater detail herein. These particlesmay include other materials such as polymers (e.g., synthetic polymers(e.g., PEG, PLGA), natural polymers (e.g., phospholipids)). In certainembodiments, the APPL is mixed with one or more agents (e.g.,cholesterol) and/or one or more other materials (e.g., polymers).

In certain embodiments, the diameter of the particles range from between1 micrometer to 1,000 micrometers. In certain embodiments, the diameterof the particles range from between from 1 micrometer to 100micrometers. In certain embodiments, the diameter of the particles rangefrom between from 1 micrometer to 10 micrometers. In certainembodiments, the diameter of the particles range from between from 10micrometer to 100 micrometers. In certain embodiments, the diameter ofthe particles range from between from 100 micrometer to 1,000micrometers. In certain embodiments, the particles range from 1-5micrometers. In certain embodiments, the diameter of the particles rangefrom between 1 nm to 1,000 nm. In certain embodiments, the diameter ofthe particles range from between from 1 nm to 100 nm. In certainembodiments, the diameter of the particles range from between from 1 nmto 10 nm. In certain embodiments, the diameter of the particles rangefrom between from 10 nm to 100 nm. In certain embodiments, the diameterof the particles range from between from 100 nm to 1,000 nm. In certainembodiments, the particles range from 1-5 nm. In certain embodiments,the diameter of the particles range from between 1 pm to 1,000 pm. Incertain embodiments, the diameter of the particles range from betweenfrom 1 pm to 100 pm. In certain embodiments, the diameter of theparticles range from between from 1 pm to 10 pm. In certain embodiments,the diameter of the particles range from between from 10 pm to 100 pm.In certain embodiments, the diameter of the particles range from betweenfrom 100 pm to 1,000 pm. In certain embodiments, the particles rangefrom 1-5 pm.

The particles may be prepared using any method known in this art. Theseinclude, but are not limited to, spray drying, single and doubleemulsion solvent evaporation, solvent extraction, phase separation,simple and complex coacervation, and other methods well known to thoseof ordinary skill in the art. In certain embodiments, methods ofpreparing the particles are the double emulsion process and spraydrying. The conditions used in preparing the particles may be altered toyield particles of a desired size or property (e.g., hydrophobicity,hydrophilicity, external morphology, “stickiness”, shape, etc.). Themethod of preparing the particle and the conditions (e.g., solvent,temperature, concentration, air flow rate, etc.) used may also depend onthe agent being encapsulated and/or the composition of the matrix.

Methods developed for making particles for delivery of encapsulatedagents are described in the literature. See, e.g., Doubrow, M., Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release 5:13-22,1987; Mathiowitz et al., Reactive Polymers 6:275-283, 1987; Mathiowitzet al., J. Appl. Polymer Sci. 35:755-774, 1988.

If the particles prepared by any of the above methods have a size rangeoutside of the desired range, the particles can be sized, for example,using a sieve. The particle may also be coated. In certain embodiments,the particles are coated with a targeting agent. In other embodiments,the particles are coated to achieve desirable surface properties (e.g.,a particular charge).

Micelles and Liposomes

The present invention further contemplates use of APPLs in thepreparation of micelles or liposomes. Any agent may be further includedin a micelle or liposome. Micelles and liposomes are particularly usefulin delivering hydrophobic agents such as hydrophobic small molecules.When the micelle or liposome is complexed with (e.g., encapsulates orcovers) a polynucleotide it is also referred to as a “lipoplex.” Manytechniques for preparing micelle and liposomes are known in the art, andany such method may be used with an APPL to make micelles and liposomes.

In certain embodiments, liposomes are formed through spontaneousassembly. In other embodiments, liposomes are formed when thin lipidfilms or lipid cakes are hydrated and stacks of lipid crystallinebilayers become fluid and swell. The hydrated lipid sheets detach duringagitation and self-close to form large, multilamellar vesicles (LMV).This prevents interaction of water with the hydrocarbon core of thebilayers at the edges. Once these particles have formed, reducing thesize of the particle can be modified through input of sonic energy(sonication) or mechanical energy (extrusion). See, e.g., Walde, P.“Preparation of Vesicles (Liposomes)” In Encylopedia of Nanoscience andNanotechnology; Nalwa, H. S. Ed. American Scientific Publishers: LosAngeles, 2004; Vol. 9, pp. 43-79; Szoka et al., “Comparative Propertiesand Methods of Preparation of Lipid Vesicles (Liposomes)” Ann. Rev.Biophys. Bioeng. 9:467-508, 1980; each of which is incorporated herein.The preparation of lipsomes involves preparing the APPL for hydration,hydrating the APPL with agitation, and sizing the vesicles to achieve ahomogenous distribution of liposomes. APPLs are first dissolved in anorganic solvent to assure a homogeneous mixture of the APPL. The solventis then removed to form a polymer-derived film. This polymer-derivedfilm is thoroughly dried to remove residual organic solvent by placingthe vial or flask on a vacuum pump overnight. Hydration of thepolymer-derived film is accomplished by adding an aqueous medium andagitating the mixture. Disruption of LMV suspensions using sonic energytypically produces small unilamellar vesicles (SUV) with diameters inthe range of 15-50 nm. Lipid extrusion is a technique in which alipid/polymer suspension is forced through a polycarbonate filter with adefined pore size to yield particles having a diameter near the poresize of the filter used. Extrusion through filters with 100 nm porestypically yields large, unilamellar polymer-derived vesicles (LUV) witha mean diameter of 120-140 nm. In certain embodiments, the amount ofAPPL in the liposome ranges from 30-80 mol %, in certain embodiments40-70 mol %, and in certain embodiments 60-70 mol %. In certainembodiments, the APPL employed further complexes an agent, such as DNAand RNA. In such embodiments, the application of the liposome is thedelivery of polynucleotides.

The following scientific papers described other methods for preparingliposomes and micelles: Narang et al., “Cationic Lipids with IncreasedDNA Binding Affinity for Nonviral Gene Transfer in Dividing andNondividing Cells” Bioconjugate Chem. 16:156-68, 2005; Hofland et al.,“Formation of stable cationic lipid/DNA complexes for gene transfer”Proc. Natl. Acad. Sci. USA 93:7305-7309, July 1996; Byk et al.,“Synthesis, Activity, and Structure-Activity Relationship Studies ofNovel Cationic Lipids for DNA Transfer” J. Med. Chem. 41(2):224-235,1998; Wu et al., “Cationic Lipid Polymerization as a Novel Approach forConstructing New DNA Delivery Agents” Bioconjugate Chem. 12:251-57,2001; Lukyanov et al., “Micelles from lipid derivatives of water-solublepolymers as delivery systems for poorly soluble drugs” Advanced DrugDelivery Reviews 56:1273-1289, 2004; Tranchant et al., “Physicochemicaloptimisation of plasmid delivery by cationic lipids” J. Gene Med.6:S24-S35, 2004; van Balen et al., “Liposome/Water Lipophilicity:Methods, Information Content, and Pharmaceutical Applications” MedicinalResearch Rev. 24(3):299-324, 2004.

Treatment Methods

It is estimated that over 10,000 human diseases are caused by geneticdisorders, which are abnormalities in genes or chromosomes. See, e.g.,McClellan, J. and M. C. King, Genetic heterogeneity in human disease.Cell. 141(2): p. 210-7; Leachman, S. A., et al., Therapeutic siRNAs fordominant genetic skin disorders including pachyonychia congenita. JDermatol Sci, 2008. 51(3): p. 151-7. Many of these diseases are fatal,such as cancer, severe hypercholesterolemia, and familial amyloidoticpolyneuropathy. See, e.g., Frank-Kamenetsky, M., et al., TherapeuticRNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents andLDL cholesterol in nonhuman primates. Proc Natl Acad Sci USA, 2008.105(33): p. 11915-20; Coelho, T., Familial amyloid polyneuropathy: newdevelopments in genetics and treatment. Curr Opin Neurol, 1996. 9(5): p.355-9. Since the discovery of gene expression silencing via RNAinterference (RNAi) by Fire and Mello (Fire, A., et al., Potent andspecific genetic interference by double-stranded RNA in Caenorhabditiselegans. Nature, 1998. 391(6669): p. 806-11), there has been extensiveeffort toward developing therapeutic applications for RNAi in humans.See, e.g., Davis, M. E., The first targeted delivery of siRNA in humansvia a self-assembling, cyclodextrin polymer-based nanoparticle: fromconcept to clinic. Mol Pharm, 2009. 6(3): p. 659-68; Whitehead, K. A.,R. Langer, and D. G. Anderson, Knocking down barriers: advances in siRNAdelivery. Nat. Rev. Drug Discovery, 2009. 8(2): p. 129-138; Tan, S. J.,et al., Engineering Nanocarriers for siRNA Delivery. Small. 7(7): p.841-56; Castanotto, D. and J. J. Rossi, The promises and pitfalls ofRNA-interference-based therapeutics. Nature, 2009. 457(7228): p. 426-33;Chen, Y. and L. Huang, Tumor-targeted delivery of siRNA by non-viralvector: safe and effective cancer therapy. Expert Opin Drug Deliv, 2008.5(12): p. 1301-11; Weinstein, S. and D. Peer, RNAi nanomedicines:challenges and opportunities within the immune system. Nanotechnology.21(23): p. 232001; Fenske, D. B. and P. R. Cullis, Liposomalnanomedicines. Expert Opin Drug Deliv, 2008. 5(1): p. 25-44; and Thiel,K. W. and P. H. Giangrande, Therapeutic applications of DNA and RNAaptamers. Oligonucleotides, 2009. 19(3): p. 209-22. Currently, there aremore than 20 clinical trials ongoing or completed involving siRNAtherapeutics, which have shown promising results for the treatment ofvarious diseases. See, e.g., Burnett, J. C., J. J. Rossi, and K.Tiemann, Current progress of siRNA/shRNA therapeutics in clinicaltrials. Biotechnol J. 6(9): p. 1130-46. However, the efficient and safedelivery of siRNA is still a key challenge in the development of siRNAtherapeutics. See, e.g., Juliano, R., et al., Biological barriers totherapy with antisense and siRNA oligonucleotides. Mol Pharm, 2009.6(3): p. 686-95.

Thus, in another aspect, provided are methods of using APPLs, e.g., forthe treatment of a disease, disorder or condition from which a subjectsuffers. It is contemplated that APPLs will be useful in the treatmentof a variety of diseases, disorders, or conditions, especially a systemfor delivering agents useful in the treatment of that particulardisease, disorder, or condition. “Disease,” “disorder,” and “condition”are used interchangeably herein. In certain embodiments, the disease,disorder or condition from which a subject suffers is caused by anabnormality in a gene or chromosome of the subject.

For example, in one embodiment, provided is a method of treatingdisease, disorder, or condition from which a subject suffers, comprisingadministering to a subject in need thereof an effective amount of acomposition comprising an APPL, or salt thereof. Exemplary disease,disorder, or conditions contemplated include, but are not limited to,proliferative disorders, inflammatory disorders, autoimmune disorders,painful conditions, liver diseases, and amyloid neuropathies.

As used herein, an “active ingredient” is any agent which elicits thedesired biological response. For example, the APPL may be the activeingredient in the composition. Other agents, e.g., therapeutic agents,as described herein may also be classified as an active ingredient. Incertain embodiments, the composition further comprises, in addition tothe APPL, a therapeutic agent useful in treating the disease, disorder,or condition. In certain embodiments, the APPL encapsulates the other(therapeutic) agent. In certain embodiments, the APPL and the other(therapeutic) agent form a particle (e.g., a nanoparticle, amicroparticle, a micelle, a liposome, a lipoplex).

In certain embodiments, the condition is a proliferative disorder and,in certain embodiments, the composition further includes an anti-canceragent. Exemplary proliferative diseases include, but are not limited to,tumors, benign neoplasms, pre-malignant neoplasms (carcinoma in situ),and malignanat neoplasms (cancers).

Exemplary cancers include, but are not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing's sarcoma, eye cancer (e.g., intraocular melanoma,retinoblastoma), familiar hypereosinophilia, gall bladder cancer,gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromaltumor (GIST), head and neck cancer (e.g., head and neck squamous cellcarcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC),throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngealcancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemiasuch as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL),acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., “Waldenström's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphomalleukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemiallymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocyticleukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilicsyndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis(NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g.,gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoidtumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma,pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer(e.g., Paget's disease of the penis and scrotum), pinealoma, primitiveneuroectodermal tumor (PNT), prostate cancer (e.g., prostateadenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer,skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g.,appendix cancer), soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous glandcarcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g.,seminoma, testicular embryonal carcinoma), thyroid cancer (e.g.,papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC),medullary thyroid cancer), urethral cancer, vaginal cancer and vulvarcancer (e.g., Paget's disease of the vulva).

Anti-cancer agents encompass biotherapeutic anti-cancer agents as wellas chemotherapeutic agents.

Exemplary biotherapeutic anti-cancer agents include, but are not limitedto, interferons, cytokines (e.g., tumor necrosis factor, interferon α,interferon γ), vaccines, hematopoietic growth factors, monoclonalserotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1,2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) andantibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab),ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR(tositumomab)).

Exemplary chemotherapeutic agents include, but are not limited to,anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRHagonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamideand bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA),phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A(2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide,trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas(e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide),platinum containing compounds (e.g. cisplatin, carboplatin,oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine,and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalentsuch as nanoparticle albumin-bound paclitaxel (ABRAXANE),docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin),polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex,CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxelbound to the erbB2-recognizing peptide EC-1), and glucose-conjugatedpaclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate;docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors(e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMPdehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin,and EICAR), ribonuclotide reductase inhibitors (e.g. hydroxyurea anddeferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine,doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosineanalogs (e.g. cytarabine (ara C), cytosine arabinoside, andfludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylationinhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline(e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin,idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDRinhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin),imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g.,axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™,AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®),gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib(TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272),nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474),vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab(AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab(VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib(NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumabozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765,AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523,PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/orXL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTORinhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus(RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235(Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502(Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)),oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed,cyclophosphamide, dacarbazine, procarbizine, prednisolone,dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,methopterin, porfiromycin, melphalan, leurosidine, leurosine,chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,aminopterin, and hexamethyl melamine.

In certain embodiments, the condition is an inflammatory disorder and,in certain embodiments, the composition further includes ananti-inflammatory agent. The term “inflammatory disorder” refers tothose diseases, disorders or conditions that are characterized by signsof pain (dolor, from the generation of noxious substances and thestimulation of nerves), heat (calor, from vasodilatation), redness(rubor, from vasodilatation and increased blood flow), swelling (tumor,from excessive inflow or restricted outflow of fluid), and/or loss offunction (functio laesa, which can be partial or complete, temporary orpermanent. Inflammation takes on many forms and includes, but is notlimited to, acute, adhesive, atrophic, catarrhal, chronic, cirrhotic,diffuse, disseminated, exudative, fibrinous, fibrosing, focal,granulomatous, hyperplastic, hypertrophic, interstitial, metastatic,necrotic, obliterative, parenchymatous, plastic, productive,proliferous, pseudomembranous, purulent, sclerosing, seroplastic,serous, simple, specific, subacute, suppurative, toxic, traumatic,and/or ulcerative inflammation.

Exemplary inflammatory disorders include, but are not limited to,inflammation associated with acne, anemia (e.g., aplastic anemia,haemolytic autoimmune anaemia), asthma, arteritis (e.g., polyarteritis,temporal arteritis, periarteritis nodosa, Takayasu's arteritis),arthritis (e.g., crystalline arthritis, osteoarthritis, psoriaticarthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis andReiter's arthritis), ankylosing spondylitis, amylosis, amyotrophiclateral sclerosis, autoimmune diseases, allergies or allergic reactions,atherosclerosis, bronchitis, bursitis, chronic prostatitis,conjunctivitis, Chagas disease, chronic obstructive pulmonary disease,cermatomyositis, diverticulitis, diabetes (e.g., type I diabetesmellitus, type 2 diabetes mellitus), a skin condition (e.g., psoriasis,eczema, burns, dermatitis, pruritus (itch)), endometriosis,Guillain-Barre syndrome, infection, ischaemic heart disease, Kawasakidisease, glomerulonephritis, gingivitis, hypersensitivity, headaches(e.g., migraine headaches, tension headaches), ileus (e.g.,postoperative ileus and ileus during sepsis), idiopathicthrombocytopenic purpura, interstitial cystitis (painful bladdersyndrome), gastrointestinal disorder (e.g., selected from peptic ulcers,regional enteritis, diverticulitis, gastrointestinal bleeding,eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis,eosinophilic gastritis, eosinophilic gastroenteritis, eosinophiliccolitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, orits synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn'sdisease, ulcerative colitis, collagenous colitis, lymphocytic colitis,ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminatecolitis) and inflammatory bowel syndrome (IBS)), lupus, multiplesclerosis, morphea, myeasthenia gravis, myocardial ischemia, nephroticsyndrome, pemphigus vulgaris, pernicious aneaemia, peptic ulcers,polymyositis, primary biliary cirrhosis, neuroinflammation associatedwith brain disorders (e.g., Parkinson's disease, Huntington's disease,and Alzheimer's disease), prostatitis, chronic inflammation associatedwith cranial radiation injury, pelvic inflammatory disease, reperfusioninjury, regional enteritis, rheumatic fever, systemic lupuserythematosus, schleroderma, scierodoma, sarcoidosis,spondyloarthopathies, Sjogren's syndrome, thyroiditis, transplantationrejection, tendonitis, trauma or injury (e.g., frostbite, chemicalirritants, toxins, scarring, burns, physical injury), vasculitis,vitiligo and Wegener's granulomatosis.

In certain embodiments, the inflammatory disorder is inflammationassociated with a proliferative disorder, e.g., inflammation associatedwith cancer.

In certain embodiments, the condition is an autoimmune disorder and, incertain embodiments, the composition further includes animmunomodulatory agent. Exemplary autoimmune disorders include, but arenot limited to, arthritis (including rheumatoid arthritis,spondyloarthopathies, gouty arthritis, degenerative joint diseases suchas osteoarthritis, systemic lupus erythematosus, Sjogren's syndrome,ankylosing spondylitis, undifferentiated spondylitis, Behcet's disease,haemolytic autoimmune anaemias, multiple sclerosis, amyotrophic lateralsclerosis, amylosis, acute painful shoulder, psoriatic, and juvenilearthritis), asthma, atherosclerosis, osteoporosis, bronchitis,tendonitis, bursitis, skin condition (e.g., psoriasis, eczema, burns,dermatitis, pruritus (itch)), enuresis, eosinophilic disease,gastrointestinal disorder (e.g., selected from peptic ulcers, regionalenteritis, diverticulitis, gastrointestinal bleeding, eosinophilicgastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilicgastritis, eosinophilic gastroenteritis, eosinophilic colitis),gastritis, diarrhea, gastroesophageal reflux disease (GORD, or itssynonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease,ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemiccolitis, diversion colitis, Behcet's syndrome, indeterminate colitis)and inflammatory bowel syndrome (IBS)), and disorders ameliorated by agastroprokinetic agent (e.g., ileus, postoperative ileus and ileusduring sepsis; gastroesophageal reflux disease (GORD, or its synonymGERD); eosinophilic esophagitis, gastroparesis such as diabeticgastroparesis; food intolerances and food allergies and other functionalbowel disorders, such as non-ulcerative dyspepsia (NUD) and non-cardiacchest pain (NCCP, including costo-chondritis)).

In certain embodiments, the condition is a painful condition and, incertain embodiments, the composition further includes an analgesicagent. A “painful condition” includes, but is not limited to,neuropathic pain (e.g., peripheral neuropathic pain), central pain,deafferentiation pain, chronic pain (e.g., chronic nociceptive pain, andother forms of chronic pain such as post-operative pain, e.g., painarising after hip, knee, or other replacement surgery), pre-operativepain, stimulus of nociceptive receptors (nociceptive pain), acute pain(e.g., phantom and transient acute pain), noninflammatory pain,inflammatory pain, pain associated with cancer, wound pain, burn pain,postoperative pain, pain associated with medical procedures, painresulting from pruritus, painful bladder syndrome, pain associated withpremenstrual dysphoric disorder and/or premenstrual syndrome, painassociated with chronic fatigue syndrome, pain associated with pre-termlabor, pain associated with withdrawal symptoms from drug addiction,joint pain, arthritic pain (e.g., pain associated with crystallinearthritis, osteoarthritis, psoriatic arthritis, gouty arthritis,reactive arthritis, rheumatoid arthritis or Reiter's arthritis),lumbosacral pain, musculo-skeletal pain, headache, migraine, muscleache, lower back pain, neck pain, toothache, dental/maxillofacial pain,visceral pain and the like. One or more of the painful conditionscontemplated herein can comprise mixtures of various types of painprovided above and herein (e.g. nociceptive pain, inflammatory pain,neuropathic pain, etc.). In some embodiments, a particular pain candominate. In other embodiments, the painful condition comprises two ormore types of pains without one dominating. A skilled clinician candetermine the dosage to achieve a therapeutically effective amount for aparticular subject based on the painful condition.

In certain embodiments, the painful condition is inflammatory pain. Incertain embodiments, the painful condition (e.g., inflammatory pain) isassociated with an inflammatory disorder and/or an autoimmune disorder.

In certain embodiments, the condition is a liver disease and, in certainembodiments, the composition further includes an agent useful intreating liver disease. Exemplary liver diseases include, but are notlimited to, drug-induced liver injury (e.g., acetaminophen-induced liverinjury), hepatitis (e.g., chronic hepatitis, viral hepatitis,alcohol-induced hepatitis, autoimmune hepatitis, steatohepatitis),non-alcoholic fatty liver disease, alcohol-induced liver disease (e.g.,alcoholic fatty liver, alcoholic hepatitis, alcohol-related cirrhosis),hypercholesterolemia (e.g., severe hypercholesterolemia),transthyretin-related hereditary amyloidosis, liver cirrhosis, livercancer, primary biliary cirrhosis, cholestatis, cystic disease of theliver, and primary sclerosing cholangitis. In certain embodiments theliver disease is associated with inflammation.

In certain embodiments, the condition is a familial amyloid neuropathyand, in certain embodiments, the composition further includes an agentuseful in a familial amyloid neuropathy.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or othernon-human animals, for example mammals [e.g., primates (e.g., cynomolgusmonkeys, rhesus monkeys); and commercially relevant mammals such asmice, rats, hampsters, cattle, pigs, horses, sheep, goats, cats, and/ordogs] and birds (e.g., commercially relevant birds such as chickens,ducks, geese, and/or turkeys). In certain embodiments, the subject is anon-human animal. The non-human animal may be a male or female and atany stage of development. A non-human animal may be a transgenic animal.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject is suffering from the specified disease, disorder or condition,which reduces the severity of the disease, disorder or condition, orretards or slows the progression of the disease, disorder or condition(“therapeutic treatment”), and also contemplates an action that occursbefore a subject begins to suffer from the specified disease, disorderor condition (“prophylactic treatment”).

In general, the “effective amount” of an active ingredient refers to anamount sufficient to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof a compound of the invention may vary depending on such factors as thedesired biological endpoint, the pharmacokinetics of the activeingredient, the disease being treated, the mode of administration, andthe age, health, and condition of the subject. An effective amountencompasses therapeutic and prophylactic treatment.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of an active ingredient is an amount sufficient toprovide a therapeutic benefit in the treatment of a disease, disorder orcondition, or to delay or minimize one or more symptoms associated withthe disease, disorder or condition. A therapeutically effective amountof an active ingredient means an amount of the active ingredient, aloneor in combination with other agents or therapies, which provides atherapeutic benefit in the treatment of the disease, disorder orcondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of disease or condition, or enhances the therapeutic efficacy ofanother therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of an active ingredient is an amount sufficient toprevent a disease, disorder or condition, or one or more symptomsassociated with the disease, disorder or condition, or prevent itsrecurrence. A prophylactically effective amount of an active ingredientmeans an amount of the active ingredient, alone or in combination withother agents or therapies, which provides a prophylactic benefit in theprevention of the disease, disorder or condition. The term“prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

The active ingredient may be administered in such amounts, time, androute deemed necessary in order to achieve the desired result. The exactamount of the active ingredient will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the infection, the particular active ingredient, its mode ofadministration, its mode of activity, and the like. The activeingredient, whether the APPL itself, or the APPL in combination with anagent, is preferably formulated in dosage unit form for ease ofadministration and uniformity of dosage. It will be understood, however,that the total daily usage of the active ingredient will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular subjectwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the activeingredient employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific active ingredient employed; the duration of the treatment;drugs used in combination or coincidental with the specific activeingredient employed; and like factors well known in the medical arts.

The active ingredient may be administered by any route. In someembodiments, the active ingredient is administered via a variety ofroutes, including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular,transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical(as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal,enteral, sublingual; by intratracheal instillation, bronchialinstillation, and/or inhalation; and/or as an oral spray, nasal spray,and/or aerosol. In general the most appropriate route of administrationwill depend upon a variety of factors including the nature of the activeingredient (e.g., its stability in the environment of thegastrointestinal tract), the condition of the subject (e.g., whether thesubject is able to tolerate oral administration), etc.

The exact amount of an active ingredient required to achieve atherapeutically or prophylactically effective amount will vary fromsubject to subject, depending on species, age, and general condition ofa subject, severity of the side effects or disorder, identity of theparticular compound(s), mode of administration, and the like. The amountto be administered to, for example, a child or an adolescent can bedetermined by a medical practitioner or person skilled in the art andcan be lower or the same as that administered to an adult.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Amino Acid-, Peptide-, and Polypeptide-Lipids (APPL) for Drug Delivery

To address the challenges associated with delivery efficiency,specificity, and toxicity of biological agents, we developed a potentand selective siRNA delivery system with a broad therapeutic windowthrough rational design and optimization of novel amino acid-based lipidderivatives.

Previously, our group has pursued a combinatorial synthetic approach todevelop new cationic lipids (lipidoids) for siRNA delivery. See, e.g.,Akinc, A., et al., A combinatorial library of lipid-like materials fordelivery of RNAi therapeutics. Nat Biotechnol, 2008. 26(5): p. 561-9;Love Kevin, T., et al., Lipid-like materials for low-dose, in vivo genesilencing. Proc Natl Acad Sci USA. 107(5): p. 1864-9; Siegwart, D. J.,et al., Combinatorial synthesis of chemically diverse core-shellnanoparticles for intracellular delivery. Proc Natl Acad Sci USA.108(32): p. 12996-3001. A number of these compounds have shownsignificant silencing effects in vivo. See, e.g., Leuschner, F., et al.,Therapeutic siRNA silencing in inflammatory monocytes in mice. NatBiotechnol. 29(11): p. 1005-10. Prior studies have identified keychemical and structural features and formulation methods for thedevelopment of new materials. See, e.g., Akinc, A., et al., Developmentof lipidoid-siRNA formulations for systemic delivery to the liver. MolTher, 2009. 17(5): p. 872-9; Akinc, A., et al., Targeted delivery ofRNAi therapeutics with endogenous and exogenous ligand-based mechanisms.Mol Ther. 18(7): p. 1357-64; Semple, S. C., et al., Rational design ofcationic lipids for siRNA delivery. Nat Biotechnol. 28(2): p. 172-6. Forexample, active compounds possess 12 or more carbons in tail length andmultiple tails. See, e.g., Love Kevin, T., et al., Lipid-like materialsfor low-dose, in vivo gene silencing. Proc Natl Acad Sci USA. 107(5): p.1864-9. In order to improve efficacy, tissue and cell-type selectivity,and tolerability, new chemical scaffolds need to be designed andinvestigated.

Amino acids are natural building blocks of peptides and proteins innature. Amino acid derivatives can be metabolized by the human body;therefore, these materials are likely well tolerated and safe astherapeutics. Additionally, peptides play significant roles in membranetransport, endogenous cellular signaling and trafficking pathways, andoffer tremendous potential in leveraging such interactions to enhancethe delivery efficiency of systems which incorporate peptide moieties.Because of their significant physiological functions and safety inhumans, amino acid-based materials, such as insulin and trastuzumab,have been widely applied as supplements and therapeutic medicines in theclinic for diverse diseases. Studies have shown that it is feasible toapply amino acid-derivatives for gene delivery or siRNA delivery. See,e.g., Prata, C. A., et al., Lipophilic peptides for gene delivery.Bioconjug Chem, 2008. 19(2): p. 418-20; Adami, R. C., et al., An aminoacid-based amphoteric liposomal delivery system for systemicadministration of siRNA. Mol Ther. 19(6): p. 1141-51; Margus, H., K.Padari, and M. Pooga, Cell-penetrating peptides as versatile vehiclesfor oligonucleotide delivery. Mol Ther. 20(3): p. 525-33. Combining theadvantages of both natural properties of amino acids and structuralfeatures of lipidoids, we applied a strategy of structural optimizationthrough an iterative screening process and rationally designed a seriesof amino acid-based lipid derivatives. We report the design, synthesis,and biological evaluation of this new series of amino acid-based lipidderivatives. This efficient and rational strategy yielded a leadmaterial cKK-E12. We systematically investigated its deliveryefficiency, tissue and cell-type selectivity, tolerability, andmechanism of action. Current results demonstrate that this deliverysystem is a novel platform for efficient, selective, and safe deliveryof siRNA, which shows great potential for the treatment of variousdiseases.

General Methods Method 1. Preparation of Compounds of Formula (I)-(III).Conjugation to Formula (i).

A mixture of amino acids, peptides or polypeptides and the conjugatingreagent (an epoxide, thiirane, or aziridine) (a ratio of 1.5:1 to 3:1conjugating reagent to amine) in EtOH was irradiated in the microwaveoven at 150° C. for 5 h. The reaction mixture was purified by flashcolumn chromatography. If amino acids, peptides or polypeptides were insalt form, triethylamine was added to the solution and stirred for 30minutes at room temperature before irradiation.

Method 2. Preparation of Compounds of Formula (I)-(III). Conjugation toFormula (ii).

A mixture of amino acids, peptides or polypeptides and conjugatingreagent (acrylate or acrylamide) (a ratio of 1.5:1 to 3:1 acrylates orconjugating reagent to amine) in ethanol (EtOH), isopropanol (iPrOH), oracetonitrile was heated to 90° C. and stirred for 2 hours to 2 days. Thereaction solution was concentrated with silica gel and purified withflash column chromatography.

Method 3. Preparation of Compounds of Formula (I)-(III). Conjugation toFormula (iii).

To a solution of amino acids, peptides or polypeptides and conjugatingreagent (aldehyde) (a ratio of 1.5:1 to 3:1 aldehydes to amine) in THFwas added sodium triacetoxyborohydride (NaBH(OAc)₃) at rt. The reactionmixture was stirred for 3 d at rt. The reaction solution wasconcentrated with silica gel and purified with flash columnchromatography.

Method 4. Preparation of Compounds of Formula (IV)

Compounds of Formula (IV) may be prepared via condensation of a1,2-diamine with an activated oxalic acid, wherein X¹ is a leavinggroup, e.g., bromo, chloro, or iodo, to provide the cyclized product.Groups of formula (i), (ii), or (iii), may be installed aftercyclization, e.g., for example, via addition to an amino side chainsubstituent of R¹, or to imino nitrogen groups R^(Q). Other groups onthe scaffold, e.g., R² groups, may be installed prior to cyclization.For example, R² may be a group of the formula (i), (ii), or (iii)installed prior to cyclization.

Method 5. Preparation of Compounds of Formula (V)

Compounds of Formula (V), and (VI) may be prepared via condensation of a1,1-diamine with an activated malonic acid, wherein X¹ is a leavinggroup, e.g., bromo, chloro, or iodo, to provide the cyclized product.Groups of formula (i), (ii), or (iii), may be installed aftercyclization, e.g., for example, via addition to an amino side chainsubstituent of R¹, or to imino nitrogen groups R^(Q). Other groups onthe scaffold, e.g., R² groups, may be installed prior to cyclization.For example, R² may be a group of the formula (i), (ii), or (iii)installed prior to cyclization.

Method 6. Preparation of Compounds of Formula (VI)

Compounds of Formula (VI) may be prepared via condensation of ahydrazine with an activated succinic acid, wherein X¹ is a leavinggroup, e.g., bromo, chloro, or iodo, to provide the cyclized product.Groups of formula (i), (ii), or (iii), may be installed aftercyclization, e.g., for example, via addition to an amino side chainsubstituent of R¹, or to imino nitrogen groups R^(Q). Other groups onthe scaffold, e.g., R² groups, may be installed prior to cyclization.For example, R² may be a group of the formula (i), (ii), or (iii)installed prior to cyclization.

Exemplary Precursors

TABLE 1 Amino Acids and Esters Name, Symbol Amino acid side chain (R¹)*Amino acid or ester Arginine R

Histidine H

Lysine K

Aspartic Acid D

Glutamic Acid E

Serine S

Threonine T

Asparagine N

Glutamine Q

Cysteine C

Glycine G —H

Proline P

Alanine A —CH₃

Beta-alanine —H, H

Valine V —CH(CH₃)₂

Isoleucine I —CH(CH₃)(CH₂CH₃)

Leucine L —CH₂CH(CH₃)₂

Methionine M

Phenylalanine F

Tyrosine Y

Tryptophan W

*R⁶ and R⁷ are hydrogen in the precursor, and, upon conjugation, areindependently selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, a nitrogen protecting group when attached to anitrogen atom, an oxygen protecting group when attached to an oxygenatom, and a sulfur protecting group when attached to a sulfur atom, or agroup of formula (i), (ii), or (iii).

TABLE 2 Peptides and Polypeptides Name, Symbol Amino acid linearlysine-lysine linear K-K

cyclic lysine-lysine cyclic K-K

polylysine K-K-K

K-K-K-K As above; n = 3 K-K-K-K-K As above; n = 4 polysine K-(K)_(n)-KAs above; n = 3-12 (500-2000 g/mol) PK-500 polysine K-(K)_(n)-K Asabove; n = 6-33 (1000-5000 g/mol) PK-1000 polysine K-(K)_(n)-K As above;n = 26-102 (4000-15000 g/mol) PK4000 polysine K-(K)_(n)-K As above; n =102-204 (15000-30000 g/mol) PK-15000 polysine K-(K)_(n)-K As above; n =204-480 (30000-70000 g/mol) PK-30000 linear arginine-arginine linear R-R

cyclic arginine-arginine cyclic R-R

polyarginine (5000-15000) R-(R)_(n)-R PR-5000

linear histidine-histidine linear H-H

cyclic histidine-histidine cyclic H-H

polyhistidine (5000-25000) H-(H)_(n)-H PH-5000

linear glycine-glycine linear G-G

cyclic glycine-glycine cyclic G-G

linear arginine-lysine linear AK

linear cysteine-lysine linear CK

linear aspartic acid-lysine linear DK

linear glutamic acid-lysine linear EK

linear phenylalanine-lysine linear FK

linear glycine-lysine linear GK

linear isoleucine-lysine linear IK

linear leucine-lysine linear LK

linear methionine-lysine linear MK

linear proline-lysine linear PK

linear glutamine-lysine linear QK

linear serine-lysine linear SK

linear tryptophan-lysine linear WK

linear tyrosine-lysine linear YK

linear lysine-threonine linear KT

linear lysine-valine linear KV

TABLE 3 Conjugating reagents Name Structure E10

E11

E12

E13

E14

E15

E16

A10

A11

A12

A13

A14

O10

O11

O12

O13

O14

N10

N11

N12

N13

N14

Synthetic Procedures Example 1. Synthesis of APPLs

Schemes A-R show the general synthetic routes to APPLs of Formula (I) to(VI), of the present invention. Application of these methods generated avariety of APPLs, depicted in Tables 4 and 5.

Compounds were named by combination of the abbreviation of amino acids,aldehydes (A), acrylates (O), amides (N), or epoxides (E), and thelength of carbon chains. For example, K-E12 represents the reaction oflysine with 1,2-epoxydodecane.

TABLE 4 Rxn Method Precursor Reagent APPL*  1 1 E12 R

R-E12  2 1 E12 A

A-E12  3 1 E12 I

I-E12  4 1 E12 E

E-E12  5 1 E12 C

C-E12  6 1 E12 W

W-E12  7 1 E12 Y

Y-E12  8 1 E12 G

G-E12  9 1 E12 P

D-E12 10 1 E12 L

L-E12 11 1 E12 D

D-E12 12 1 E12 S

S-E12 13 1 E12 T

T-E12 14 1 E12 H

H-E12 15 1 E12 N

N-E12 16 1 E12 Q

Q-E12 17 1 E12 F

F-E12 18 1 E12 M

M-E12 19 1 E12 V

V-E12 20 3 A12 K

K-A12 21 3 A12 KK

KK-A12 22 1 E12 K

K-E12 23 1 E12 cyclic- KK

cKK-E12 24 1 E12 PK-500

polyK₅₀₀-E12 25 1 E12 PK-1000 As above, n = 6-33 polyK₁₀₀₀-E12 26 1 E12PK-4000 As above, n = 26-102 polyK₄₀₀₀-E12 27 1 E12 PK-15000 As above, n= 102-204 polyK₁₅₀₀₀-E12 28 1 E12 PK-30000 As above, n = 204-480polyK₃₀₀₀₀-E12 29 1 E10 cyclic- KK

cKK-E10 30 1 E14 cyclic-KK

cKK-E14 31 1 E16 cyclic-KK

cKK-E16 32 1 E11 cyclic- KK

cKK-E11 33 1 E13 cyclic- KK

cKK-E13 34 1 E15 cyclic- KK

cKK-E15 35 1 E12 Boc-Lys- OH

NBoc-K-E12 36 1 E12 H- Lys(Boc)- OMe

(side chain N-Boced) K-E12 37 2 N12 cKK

plus compounds wherein 1 tail (R^(L)) is replaced with hydrogen. cKK-N1238 2 O12 cKK

cKK-O12 39 3 A12 cKK

cKK-A12 40 1 E12 KKK

KKK-E12 41 2 A12 KKK

KKK-A12 42 3 O12 KKK

plus compounds wherein 1 tail (R^(L)) is replaced with hydrogen. KKK-O1243 3 O12 KK

plus compounds wherein 1 tail (R^(L)) is replaced with hydrogen. KK-O1244 3 N12 KKK

plus compounds wherein 1 tail (R^(L)) is replaced with hydrogen. KKK-N12*reaction may produce a mixture of two or more APPLs; also includescompounds wherein 1 or more tails (R^(L)) are replaced with hydrogen.

TABLE 5 Compd Chemical formula Calcd. Observed Tail # A-E12 C27H54NO3+440.4098 440.4336 2 C-E12 C27H54NO3S+ 472.3819 472.4303 2 D-E12C28H54NO5+ 484.3997 484.4327 2 E-E12 C29H56NO5+ 498.4153 498.4117 2F-E12 C33H58NO3+ 516.4411 516.4332 2 G-E12 C26H52NO3+ 426.3942 426.37722 H-E12 C42H80N3O4+ 690.6143 690.6016 3 I-E12 C30H60NO3+ 482.4568482.4461 2 K-E12 C42H85N2O4+ 681.6504 681.6009 3 L-E12 C30H60NO3+482.4568 482.4771 2 M-E12 C29H58NO3S+ 500.4132 500.4471 2 N-E12C40H79N2O5+ 667.5984 667.5894 3 P-E12 C17H32NO2+ 282.2428 282.2585 1Q-E12 C29H57N2O4+ 497.4313 497.4268 2 R-E12 C54H109N4O5+ 893.8392893.8400 4 S-E12 C27H54NO4+ 456.4047 456.3891 2 T-E12 C28H56NO4+470.4204 470.4186 2 V-E12 C29H58NO3+ 468.4411 468.4259 2 W-E12C35H59N2O3+ 555.4520 555.4510 2 Y-E12 C33H58NO4+ 532.436 532.4149 2cKG-E12 C32H64N3O4+ 554.4891 554.4852 2 cKT-E12 C34H68N3O5+ 598.5153598.5179 2 cYK-E12 C39H70N3O5+ 660.5310 660.5350 2 cLK-E12 C36H72N3O4+610.5517 610.5556 2 cDK-E12* C36H70N3O6+ 640.5259 640.5316 2 cMK-E12C35H70N3O4S+ 628.5082 628.5072 2 cKV-E12 C35H70N3O4+ 596.5361 596.5330 2cAK-E12 C33H66N3O4+ 568.5048 568.4992 2 cCK-E12 33H66N3O4S+ 600.4769600.6143 2 cQK-E12 C35H69N4O5+ 625.5262 625.4733 2 cPK-E12 C35H68N3O4+594.5204 594.5169 2 cFK-E12 C39H70N3O4+ 644.5361 644.5301 2 cWK-E12C41H71N4O4+ 683.5470 683.5367 2 cEK-E12 C35H68N3O6+ 626.5103 626.5053 2cIK-E12 C36H72N3O4+ 610.5517 610.5501 2 cSK-E12 C33H66N3O5+ 584.4997584.5029 2 cKK-E10 C52H105N4O6+ 881.8029 881.8042 4 cKK-E12 C60H121N4O6+993.9281 993.9224 4 cKK-E14 C68H137N4O6+ 1106.0533 1106.0709 4 cKK-E16C76H153N4O6+ 1218.1785 1218.2002 4 A-A12 C27H56NO2+ 426.4306 426.4244 2C-A12 C27H56NO2S+ 458.4026 458.3857 2 D-A12 C28H56NO4+ 470.4204 470.41882 E-A12 C29H58NO4+ 484.4360 484.4319 2 F-A12 C33H60NO2+ 502.4619502.4560 2 G-A12 C26H54NO2+ 412.4149 412.4107 2 H-A12 C30H58N3O2+492.4524 492.4503 2 I-A12 C30H62NO2+ 468.4775 468.4714 2 K-A12C54H111N2O2+ 819.8640 819.8657 4 L-A12 C30H62NO2+ 468.4775 468.4752 2M-A12 C29H60NO2S+ 486.4339 486.4318 2 N-A12 C28H57N2O3+ 469.4364469.4328 2 P-A12 C17H34NO2+ 284.2584 284.2512 1 Q-A12 C29H59N2O3+483.4520 483.4543 2 R-A12 C42H87N4O2+ 679.6824 679.6783 3 S-A12C27H56NO3+ 442.4255 442.4225 2 T-A12 C28H58NO3+ 456.4411 456.4398 2V-A12 C29H60NO2+ 454.4619 454.4544 2 W-A12 C35H61N2O2+ 541.4728 541.47242 Y-A12 C33H60NO3+ 518.4568 518.4543 2 KK-A12 C84H171N4O3+ 1284.33461284.3458 6 KKK-A12 C114H231N6O4+ 1748.8051 1748.8340 8 cKK-A12C60H121N4O2+ 929.9484 929.9445 4 A-O12 C18H36NO4+ 330.2639 330.2582 1C-O12 C33H64NO6S+ 602.4449 602.4426 2 D-O12 C19H36NO6+ 374.2537 374.24921 E-O12 C20H38NO6+ 388.2694 388.2672 1 F-O12 C24H40NO4+ 406.2952406.2896 1 G-O12 C17H34NO4+ 316.2482 316.2423 1 H-O12 C36H66N3O6+636.4946 636.4969 2 I-O12 C21H42NO4+ 372.3108 372.3054 1 K-O12C66H127N2O10+ 1107.9485 1107.9417 4 L-O12 C21H42NO4+ 372.3108 372.3052 1M-O12 C20H40NO4S+ 390.2673 390.2628 1 N-O12 C19H37N2O5+ 373.2697373.2668 1 P-O12 C20H38NO4+ 356.2795 356.2779 1 Q-O12 C20H39N2O5+387.2853 387.2831 1 R-O12 C21H43N4O4+ 415.3279 415.3235 1 S-O12C18H36NO5+ 346.2588 346.2521 1 T-O12 C19H38NO5+ 360.2744 360.2733 1V-O12 C20H40NO4+ 358.2952 358.2905 1 W-O12 C26H41N2O4+ 445.3061 445.30101 Y-O12 C24H40NO5+ 422.2901 422.2868 1 KK-O12 C87H167N4O13+ 1476.25241476.2533 5 KKK-O12 C123H235N6O18+ 2084.7652 2084.7650 7 cKK-O12C72H137N4O10+ 1218.0329 1218.0880 4 *formation of ethyl ester. Compoundsderived from poly-L-lysine are not included.

Exemplary Compounds of Table 5:

Example 2. Alternative Synthesis of Compound 23 (cKK-E12)

Synthesis of Compound B.

Compound A (487 mg, 1.02 mmol) was charged in a 10 ml flask andtrifluoroacetic acid (TFA, 1.3 mL) was added dropwise at 0° C. Thereaction mixture was warmed to room temperature and stirred for 30 min.The solvents were evaporated under reduced pressure and the TFA salts inDMF (3.5 mL) were added dropwise to pyridine (100 mL) at 0° C. Thereaction mixture was slowly warmed to room temperature and stirred forovernight. The solvents were evaporated under reduced pressure and thewhite solid was washed with EtOAc to give pure B in 69% yield. MS: m/z525 (M+H⁺); ¹H NMR (500 MHz, DMSO, ppm): δ 1.29-1.40 (m, 8H, CH₂CH₂),1.61-1.68 (m, 4H, CH₂), 2.97 (dd, J=6.0, 12.5 Hz, 4H, NCH₂), 3.79 (br,2H, COCH), 7.22 (t, J=5.5 Hz, 2H, aromatic), 7.33-7.37 (m, 8H,aromatic), 8.10 (s, 2H, NH).

Synthesis of Compound C.

A cloudy solution of compound B (95 mg, 0.18 mmol) in 50% aceticacid/CH₂Cl₂ (6 mL) was added Pd on charcoal (10 wt %, 36.5 mg). Theblack suspension was degassed for 5 mins and hydrogen gas introduced.The reaction mixture stirred at rt overnight and was then filteredthrough a layer of Celite, which was washed several times with MeOH. Thecombined filtrates were concentrated to obtain a yellow viscous oil,which was solidified by adding EtOAc. The solid was washed by ethylacetate to yield compound C in 90% yield. MS: m/z 257 (M+H⁺); ¹H NMR(500 MHz, D₂O, ppm): δ 1.39-1.52 (m, 4H, CH₂), 1.67-1.71 (m, 4H, CH₂),1.84-1.88 (m, 4H, CH₂), 2.99 (t, J=7.5 Hz, 4H, NCH₂), 4.14 (t, J=5.0 Hz,2H, COCH).

Synthesis of Compound 23 (cKK-E12).

A mixture of compound C (169.2 mg, 0.45 mmol) and 1,2-epoxydodecane (523mg, 2.7 mmol) in EtOH was added triethylamine (182 mg, 1.8 mmol), whichwas stirred 30 mins at rt. The reaction mixture was then irradiated inthe microwave oven at 150° C. for 5 h. The mixture was purified by flashcolumn chromatography to obtain compound 23 (in 52% yield) as a lightyellow oil. MS: m/z 993 (M+H⁺); ¹H NMR (500 MHz, DMSO, ppm): δ 0.87 (t,J=7.0 Hz, 12H, CH₃), 1.21-1.39 (m, 80H, CH₂), 1.64-1.67 (m, 4H, CH₂),2.25-2.44 (m, 12H, NCH₂), 3.44 (br, 4H, CHOH), 3.79 (br, 2H, COCH), 4.21(d, J=3.0 Hz, 2H, CHOH), 4.27 (d, J=3.0 Hz, 2H, CHOH), 8.11 (br, 2H,CONH).

Example 3. Synthesis of Compound D

It is envisioned compound D can be synthesized by reaction of 23 withLawesson's reagent in dry toluene.

Example 4. Synthesis of Compound E

It is envisioned compound E can be synthesized by reaction of 23 withhydroxylamine hydrochloride or other substituted amines in methanol.

Biological Methods

siRNA Formulations

Formulation A

APPL, distearoyl phosphatidylcholine (DSPC), cholesterol andmPEG2000-DMG were solubilized in 90% ethanol at a molar ratio of50:10:38.5:1.5. The siRNA (against firefly luciferase or fVII) wassolubilized in 10 mM citrate, pH 3 buffer at a concentration of 0.4mg/nL. The ethanolic lipid solution and the aqueous siRNA solution werepumped by means of a syringe pump through a microfluidic mixing chamberto spontaneously form siRNA-containing lipid nanoparticles. Lipids werecombined with siRNA at a total lipid to siRNA ratio of 7:1 (wt:wt).These formulations were dialyzed against PBS to remove ethanol andexchange buffer.

Formulation B

APPLs were formulated with cholesterol (Sigma-Aldrich), DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine, Avanti), mPEG2000-DMG(synthesized by Alnylam), and siRNA via a microfluidic based mixingdevice See, e.g., Chen, D., et al., Rapid Discovery of PotentsiRNA-Containing Lipid Nanoparticles Enabled by Controlled MicrofluidicFormulation. J Am Chem Soc. Formulations were then dialyzed against PBSin 3,500 MWCO dialysis cassettes (Pierce) overnight. Particles werecharacterized with a modified Ribogreen assay (Invitrogen) for siRNAentrapment and dynamic light scattering (ZetaPALS, BrookhavenInstruments) for mean particle diameter. cKK-E12 formulations were madefrom cholesterol, DSPC, and mPEG2000-DMG using a similar method at amolar ratio of 50:10:38.5:1.5. This formulation afforded a particlediameter of 60-70 nm with approximately 65% siRNA entrapment.

In Vitro Luciferase Gene Silencing

HeLa cells, stably expressing firefly luciferase and Renilla luciferase,were seeded (14,000 cells/well) into each well of an opaque white96-well plate (Corning-Costar) and allowed to attach overnight in growthmedium. Growth medium was composed of 90% phenol red-free DMEM, 10% FBS,100 units/ml penicillin, 100 mg/ml streptomycin (Invitrogen). Cells weretransfected with LNPs formulated with anti-luciferase siRNA by additionof formulated particles to growth medium. Transfections were performedin quadruplicate. Cells were allowed to grow for 1 d at 37° C., 5% CO2and were then analyzed for luciferase expression. Control experimentswere performed with Lipofectamine 2000, as described by the vendor(Invitrogen). Firefly and Renilla luciferase expression was analyzedusing Dual-Glo assay kits (Promega). Luminescence was measured using aVictor3 luminometer (Perkin Elmer).

In Vivo Factor VII Gene Silencing in Mice

C57BL/6 mice (Charles River Labs) were used for siRNA silencingexperiments. Prior to injection, formulations were diluted in PBS atsiRNA concentrations (SEQ ID NO 1 (siFVII sense):5′-GGAucAucucAAGucuuAcT*T-3′; SEQ ID NO 2 (antisense):5′-(uAAGAcuuGAGAuGGAuGAuccT*T-3′) such that each mouse was administereda dose of 0.01 mL/g body-weight. Formulations were administeredintravenously via tail vein injection. After 48 or 72 h, body-weightgain/loss was measured and mice were anaesthetized by isofluoraneinhalation for blood sample collection by retroorbital eye bleed. Serumwas isolated with serum separation tubes (Falcon tubes, BectonDickinson) and Factor VII protein levels were analyzed by chromogenicassay (Biophen FVII, Aniara Corporation). A standard curve wasconstructed using samples from PBS-injected mice and relative Factor VIIexpression was determined by comparing treated groups to untreated PBScontrol.

Biodistribution Cy5.5-Labeled siRNA-cKK-E12 Formulation in Mice.

The mice mentioned above were systemically injected with formulatedCy5.5-labeled siRNA at a dose of 1 mg/kg of total siRNA. The mice weresacrificed 1 hour or 24 hours post injection; the pancreas, spleen,liver, kidneys, ovaries, uterus, heart, lungs, and thymus as well as asection of the adipose tissue and muscle tissue were then removed andimaged. The organs were examined with an Ivis imaging system fromCaliper using an excitation wavelength of 675 nm and an emissionwavelength of 720 nm. The data were processed using the Living Imagesoftware from Caliper. Signal strength of the individual organs wasnormalized against the total signal strength of all organs.

In Vitro siRNA Transfection Assay and Microscopy.

Effects of apolipoproteins were evaluated through an in vitro siRNAtransfection assay in HeLa cells as previously reported. HeLa cells,stably expressing firefly luciferase and Renilla luciferase were seededin an opaque white 96-well plate (Corning-Costar) overnight. Cells weretransfected by cKK-E12 formulated with 50 ng of firefly-specific siLucin quadruplicate. Apolipoproteins (Fitzgerald Industries) were incubatedwith cKK-E12 formulations for 5 mins before adding to cells. After 24 hincubation at 37° C., 5% CO₂, cells were analyzed for luciferaseexpression using Dual-Glo assay kits (Promega). For visualization ofcell uptake, cKK-E12 was formulated with an Alexa-Fluor 647-labeledsiRNA and incubated with Hela cells for 3 h. Cells were then fixed in 4%paraformaldehyde, permeabilized with 0.1% saponin and stained withHoescht. All images were acquired using an Opera spinning disc confocalsystem (Perkin Elmer), and the data was analyzed using Acapella Software(Perkin Elmer).

Discussion

Single amino acids were reacted with aldehydes, acrylates, and epoxidesto produce APPLs. The newly-synthesized single amino acid-based lipidderivatives were evaluated for their capacity to silence hepatic genesin mice. A validated genetic target, Factor VII (a blood clottingfactor), was selected as a silencing marker. See, e.g., Akinc, A., etal., A combinatorial library of lipid-like materials for delivery ofRNAi therapeutics. Nat Biotechnol, 2008. 26(5): p. 561-9. New lipidderivatives were formulated with cholesterol, DSPC, PEG-lipid, and siRNAvia a microfluidic based mixing technology. See, e.g., Chen, D., et al.,Rapid Discovery of Potent siRNA-Containing Lipid Nanoparticles Enabledby Controlled Microfluidic Formulation. J Am Chem Soc. Formulations thatwere instable in solution or had no siRNA entrapment were not screened.Stable formulations were injected in mice through systemicadministration at a dose of 1 mg/kg (FIG. 1). From this initialscreening, we identified that K-E12 was more potent than others. The hitrate (over 50% silencing) was one out of 60 compounds (i.e. 1.7%,including those compounds not screened due to particle instability or noentrapment of siRNA).

The enhanced potency of K-E12 led to our design of a second set oflysine-based peptide and polypeptide-lipid derivatives. Lysine-baseddipeptides were reacted with epoxides to give diketopiperizine APPLs.Microwave irradiation was utilized to produce these scaffolds, whichdramatically reduced the reaction time from 3 days to 5 hours. Inaddition, to further confirm the chemical structure and improve chemicalavailability for large-scale synthesis, an alternative synthetic routewas developed for the synthesis of cKK-E12 (Example 2). Diamine 5 wassynthesized according to the method reported previously (Bergeron, R.J., et al., Macromolecular Self-Assembly of DiketopiperazineTetrapeptides. J. Am. Chem. Soc., 1994. 116(19): p. 8479-84; Kaur, N.,et al., A Delineation of Diketopiperazine Self-Assembly Processes:Understanding the Molecular Events Involved inN-(Fumaroyl)diketopiperazine of L-Lys (FDKP) Interactions. Mol.Pharmaceutics, 2008. 5(2): p. 294-315), which reacted with1,2-epoxydodecane to afford cKK-E12. Compound (C) underwent reductiveamination or Michael addition reactions with dodecanal or dodecylacrylate to yield cKK-A12 and cKK-O12. Reactions between lysine-lysineand poly-L-lysine (molecular weight from 500-70000 g/mol) and aldehydesand acrylates were similar to those of single amino acids.

The silencing effects were next evaluated. Ten out of 43 compoundsshowed around 50% silencing at a dose of 1 mg/kg. The hit rate of thesecond set of compounds was 23%, which was over 10-fold more efficientcompared to the first set of materials. The results suggested that ouriterative screening process is an efficient strategy for identifyinglead compounds. The results from the second set also showed that epoxidederivatives were more potent than aldehyde and acrylate derivatives(such as cKK-E12 vs cKK-A12 & cKK-O12). Hit materials were furthertested at a lower dose of 0.1 mg/kg. The tail length significantlyaffects silencing and 12-14 carbon tail lengths appeared favorable(cKK-E10, -E12, -E14, & -E16). cKK-E12 was the most potent material andwas selected for further exploration.

Biodistribution Study

A biodistribution study was performed with naked Cy5.5 labeled siRNA andformulated cKK-E12. By subtracting the contribution of free siRNA in theformulation of cKK-E12, over 80% of particles were located in the liverat 1 hr and most residual siRNA was cleared by 24 hr through kidney(FIG. 2).

Effects of Apolipoproteins on Cell Uptake and Gene Silencing

Previous studies have reported that Apolipoprotein E (ApoE) was able toenhance cell uptake and gene silencing for a certain type of materials.Akinc, A., et al., Targeted delivery of RNAi therapeutics withendogenous and exogenous ligand-based mechanisms. Mol Ther. 18(7): p.1357-64. In order to test the effects of diverse apoliproteins on celluptake and gene silencing, and explore the mechanism of action,experiments were performed with cKK-E12 and 11 isoforms of ApoA, ApoB,ApoC, ApoE, and ApoH. Results in Hela cells showed that mostapolipoproteins did not affect cell viability with the exception ofApoB. ApoA, ApoC, and ApoH did not show significant effects on silencingcompared to free cKK-E12 (FIG. 3). However, four different ApoE isoformssignificantly improved luciferase silencing.

The activity of cKK-E12, cKK-A12, and cKK-O12 was compared with andwithout addition of apoE3 (apoE3 is the dominant isoform in humans. FIG.4A). Without addition of ApoE3, cKK-A12 was more potent than cKK-E12 andcKK-O12. However, with addition of ApoE3, the order of silencing effectswas cKK-E12>cKK-A12>cKK-O12, which correlated well with in vivoactivity. The results suggested that a cell assay with addition of ApoEmight be a practical and effective model for preliminary screening forliver hepatocytes silencing. In addition, the cell uptake of cKK-E12formulated with an Alexa-Fluor 647 labeled siRNA was visualized usingautomated confocal microscopy (FIG. 4B).

Other Embodiments

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, books, manuals, and other publications,all of which are incorporated herein by reference. If there is aconflict between any of the incorporated references and the instantspecification, the specification shall control. In addition, anyparticular embodiment of the present invention that falls within theprior art may be explicitly excluded from any one or more of the claims.Because such embodiments are deemed to be known to one of ordinary skillin the art, they may be excluded even if the exclusion is not set forthexplicitly herein. Any particular embodiment of the invention can beexcluded from any claim, for any reason, whether or not related to theexistence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

1-85. (canceled)
 86. A method of treating a disease, disorder, orcondition from which a subject suffers, comprising administering to asubject in need thereof an effective amount of a composition comprisingan agent and a compound of Formula (III):

or salt thereof, with the cell under conditions sufficient to facilitatedelivery of an agent into the interior of the cell; wherein: p is 1;each instance of Q is O; each instance of R¹ is independently hydrogen,alkyl, or a group of formula (iv); provided at least one instance of R¹is a group of formula:

L is alkylene; R⁶ and R⁷ are each independently hydrogen, alkyl, anitrogen protecting group, or a group of the formula (i), (ii), or(iii); each instance of R² is independently hydrogen, alkyl, a nitrogenprotecting group, or a group of the formula (i), (ii), or (iii);Formulae (i), (ii), and (iii) are:

wherein each instance of formula (i) is independently formula (i-a) orformula (i-b):

each instance of R′ is independently hydrogen or alkyl; X is O, S, orNR^(X), wherein R^(X) is hydrogen or a nitrogen protecting group; Y isO; R^(P) is hydrogen, alkyl, an oxygen protecting group when attached toan oxygen atom, a sulfur protecting group when attached to a sulfuratom, or a nitrogen protecting group when attached to a nitrogen atom;and R^(L) is C₆₋₅₀ alkyl, provided that at least one instance of R², R⁶,or R⁷ is a group of the formula (i), (ii), or (iii); wherein each alkyland alkylene is independently unsubstituted or substituted at one ormore positions with a group selected from halogen, —CN, —NO₂, —OH,—OR^(aa), —N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH,—SR^(aa), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, C₃₋₁₄ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, wherein eachalkyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 groups selected from halogen, —OH,—OR^(ee), and C₁₋₅₀alkyl; or two geminal hydrogens on a carbon atom arereplaced with the group ═O; each instance of R^(aa) is, independently,selected from C₁₋₅₀alkyl; each instance of R^(bb) is, independently,selected from hydrogen, and C₁₋₅₀ alkyl; each instance of R^(cc) is,independently, selected from C₁₋₅₀ alkyl, and C₃₋₁₀ carbocyclyl; whereinX⁻ is a counterion.
 87. The method of claim 86, wherein each instance ofR¹ is a group of formula (iv).
 88. The method of claim 86, wherein thecompound is selected from the group consisting of:

and salts thereof, wherein: R¹ is selected from the group consisting of—H, —CH₃, —CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH₂CH(CH₃)₂,

provided at least one R¹ is a group of formula:


89. The method of claim 86, wherein the compound is selected from thegroup consisting of:

and salts thereof.
 90. The method of claim 86, wherein the compound isselected from the group consisting of:

and salts thereof.
 91. The method of claim 90, wherein the compound is:

or salt thereof.
 92. The method of claim 86, wherein X is NR^(X),wherein R^(X) is hydrogen or a nitrogen protecting group.
 93. The methodof claim 86, wherein X is O.
 94. The method of claim 86, wherein thecomposition is a pharmaceutical composition.
 95. The method of claim 86,wherein the agent is an organic molecule, inorganic molecule, nucleicacid, protein, peptide, polynucleotide, targeting agent, an isotopicallylabeled chemical compound, vaccine, an immunological agent, or an agentuseful in bioprocessing.
 96. The method of claim 86, wherein the agentis a polynucleotide.
 97. The method of claim 96, wherein thepolynucleotide is RNA.
 98. The method of claim 97, wherein the RNAcarries out RNA interference.
 99. The method of claim 97, wherein theRNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA.
 100. Themethod of claim 97, wherein upon delivery of the RNA into the cell, theRNA is able to interfere with the expression of a specific gene in abiological cell.
 101. The method of claim 96, wherein the polynucleotideis DNA.
 102. The method of claim 96, wherein the polynucleotide encodesa protein or peptide.
 103. The method of claim 86, wherein the disease,disorder, or condition is selected from the group consisting ofproliferative disorders, inflammatory disorders, autoimmune disorders,painful conditions, liver diseases, and familial amyloid neuropathies.104. The method of claim 86, wherein the disease, disorder, or conditionis cancer.
 105. The method of claim 86, wherein the disease, disorder,or condition is a liver disease selected from the group consisting ofdrug-induced liver injury, hepatitis, non-alcoholic fatty liver disease,alcohol-induced liver disease, hypercholesterolemia,transthyretin-related hereditary amyloidosis, liver cirrhosis, livercancer, primary biliary cirrhosis, cholestasis, cystic disease of theliver, and primary sclerosing cholangitis.